JPH0764891B2 - Boron-containing cross-linked copolymer chromatography gel - Google Patents

Description

TECHNICAL FIELD The present invention relates to a gel for crosslinked copolymer chromatography having a hydroxyl group and a dihydroxyboronyl group. It is well known that a compound having a dihydroxyboronyl group has a property of reacting with a compound having a cis-type diol structure to form a complex, as shown in the following formula (1). Utilizing this, for example, a substance in which a dihydroxyboronyl group is immobilized on an insoluble carrier is used for separation and analysis of sugars, nucleotides, nucleic acids, catecholamines, glycoproteins and the like. The boron-containing cross-linked copolymer gel of the present invention efficiently separates these substances,
Suitable for analysis.

[Prior Art] As a carrier for immobilizing a dihydroxyboronyl group, cellulose [H. L. Weiss et al., Biochemistry (HLWeith, et.al., Biochemistry) 9 ,
4396 (1970)], agarose, dextran [USP3,91
2,595], polyacrylamide [R Duncan, et al., Anal Biochem (REDuncan, et.al., Anal.B
iochem,) 66, 532 ('75)] and other soft gels. However, soft gels such as cellulose, dextran, and polyacrylamide have remarkably low mechanical strength and break down during handling, and when packed in a column and used, liquid cannot flow at a high flow rate. Have.
It is also known that glass beads are fixed with a dihydroxyboronyl group [RE Duncan et al., Anal Biochem (REDuncan, et.al., Anal.Bioche
m.) 66, 532 ('75)].

The dihydroxyboronyl group interacts with 1,2 cisdiol compounds under alkaline conditions. Therefore, in order to separate and analyze sugars and glycoproteins as described above, it is preferable to bring them into contact with a dihydroxyboronyl group under alkaline conditions. However, glass has a drawback that it melts under alkaline conditions and is not always preferable as a carrier for immobilizing dihydroxyboronyl groups. Further, a polymer in which an aminophenyldihydroxyboronyl group is bonded to a copolymer of glycidyl methacrylate and butanediol dimethacrylate is also known (JP-A-55-66910), but the hydrophobic structure of butanediol dimethacrylate forming a skeleton is known. Due to its properties, hydrophobic adsorption may occur, which is not preferable. [Chromatography of Synthetic and Biopolymers]
Logical Polymers) vol 1, P101, ELLIS HORWOOD LTD], and polymers obtained by copolymerization of P-vinyl boric acid (P hydroxypolystyrene) and divinylbenzene are also known ( JP-A-59-223706), however, a copolymer of styrene and divinylbenzene is extremely poor in hydrophilicity and may also cause hydrophobic adsorption, which is not preferable.

[Problems to be solved by the invention]

As described above, the conventional dihydroxyboronyl group-containing polymer may have insufficient strength, may be chemically unstable, or may cause hydrophobic adsorption. For example, when separating and analyzing sugars, nucleosides, nucleotides, glycoproteins, etc., there is a possibility that the above-mentioned problems may occur.

Therefore, as a result of diligent studies, the present inventors have found that a hard gel, which is stable in a wide pH range, and has high hydrophilicity, has less nonspecific adsorption of proteins and the like, and thus has a dihydroxyboronyl group-containing cross-linked copolymer chromatography gel. The development was successful and the present invention was completed.

[Means for solving problems]

That is, the present invention, the cross-linked copolymer weight, alcoholic hydroxyl group 1.0 ~ 14.0meq / g, dihydroxyboronyl group 0.05.
The present invention relates to a boron-containing cross-linked copolymer chromatography gel having a surface area of 5 to 1000 m ² / g, a surface area of 5 to 1000 m ² / g, and an amount of water that can be retained from 0.5 to 6.0 g / g.

The boron-containing crosslinked copolymer gel (hereinafter referred to as gel) of the present invention has 1.0 to 14.0 meq / g of alcoholic hydroxyl group per weight of the crosslinked copolymer. By having a hydroxyl group in this range, the copolymer has appropriate hydrophilicity and does not exhibit non-specific adsorption for proteins and the like. If the amount of hydroxyl groups is less than 1.0 meq / g, hydrophobic interaction with proteins may occur, which is not preferable. If the amount of hydroxyl groups is more than 14.0 meq / g, the strength of the copolymer may be insufficient, which is not preferable. The amount of alcoholic hydroxyl group is preferably 2.0 per copolymer weight.
~ 14.0 meq / g.

The amount of hydroxyl groups can be determined, for example, by the method described in JP-A-57-30945. In the copolymer of the present invention, it is preferable to measure the amount of hydroxyl groups after the boron is cleaved and released from the carbon chain using, for example, an aqueous hydrogen peroxide solution. In that case, since a hydroxyl group is derived from the carbon atom to which boron was bound, by subtracting the amount of boric acid obtained separately from the amount of the hydroxyl group obtained by the above method, the hydroxyl group of the copolymer The quantity can be calculated. In the method disclosed in JP-A-57-30945, a polymer is reacted with acetic anhydride, and the amount of hydroxyl groups is determined from the amount of acetic anhydride reacted.

Therefore, if an amino group is present in the copolymer,
The amount of hydroxyl groups can be determined by subtracting the amount of amino groups from the amount of reacted acetic anhydride. The amount of amino groups can be determined separately by titration or the like.

The gel of the present invention has a dihydroxyboronyl group of 0.05 to 5.0 m.
Included in the range of eq / g. The presence of the dihydroxyboronyl group in this range shows a suitable interaction with a water-soluble substance having a cis-1,2 diol structure, for example, in an aqueous solution. The amount of the dihydroxyboronyl group can be determined, for example, by allowing a polymer having a dihydroxyboronyl group and sorbitol to coexist in an aqueous solvent and titrating with an alkali [New Experimental Chemistry Course, Analytical Chemistry I, P7.
7, Maruzen). It can also be obtained by treating a polymer having a dihydroxyboronyl group with hydrogen peroxide and determining the amount of released boron by the above-mentioned titration method, atomic absorption method, luminescence method or the like.

The gel of the present invention has a specific surface area of 5 to 1000 m ² / g. Soft gels such as Sephadex shrink when dried, and because the pores are lost, they show only a specific surface area of 1 m ² / g or less, but the gel of the present invention is hard and has permanent pores, so the pores when dried are Although it contracts to some extent, it maintains most of its swelled state.

The specific surface area can be determined, for example, by the method described in JP-A-57-30945.

The water retention capacity of the gel of the present invention (hereinafter referred to as W _R ) is 0.5 to 6.0 g / g
Is suitably in the range of 1.0 to 5.0 g / g
Is the range. W _R is the amount of water that can be contained in the gel when the gel is equilibrated with water, and is expressed as a value per dry weight of the cross-linked copolymer (gel). In other words, W _R is a measure of the amount of pores in the gel. When W _R becomes large, the weight of the gel itself, that is, the portion forming the skeleton per unit volume of the gel in water relatively decreases. Therefore W _R
If is too large, the mechanical strength of the gel in water decreases. If R _R is too small, the amount of pores effective for separation decreases, so the separation ability decreases. Therefore, it is preferable that W _R is in an appropriate range. W _R can be obtained by the method described in JP-A-57-30945, for example.

The gel of the present invention may have any shape such as a sphere, a lump, or a film, but when it is packed in a column and used, the sphere is preferable, and in that case, the particle diameter is preferably in the range of 3 to 1000 μm.

The proportion of the crosslinkable monomer units in all the monomer units forming the gel of the present invention is such that X in the formula (2) is in the range of 0.2 ≦ X ≦ 0.4.

(However, a is a mole fraction of the monomer unit excluding the crosslinkable monomer unit in the copolymer, b is a mole fraction of the crosslinkable monomer unit, and n is one molecule of the crosslinkable monomer. It represents the number of functional groups such as vinyl groups that can be polymerized and is used as a packing material for high performance liquid chromatography with the copolymer of the present invention. Examples of the skeleton of the gel of the present invention include crosslinked polymers having a hydroxyl group. This hydroxyl group is derived from a vinyl alcohol unit. Particularly preferred examples include those in which vinyl alcohol units are crosslinked with triallyl isocyanurate units.

Another example of the crosslinked copolymer gel of the present invention is polyvinyl alcohol crosslinked with epihalohydrin or bisepoxide. The skeleton of the copolymer of the present invention can be obtained, for example, by copolymerizing vinyl acetate and triallyl isocyanurate and then saponifying as described in JP-A-57-30945.

The gel of the present invention comprises, for example, introducing a carboxymethyl group into a hydroxyl group of a cross-linked copolymer having a hydroxyl group as described above by a known method, then converting the carboxymethyl group into an azide, and then reacting with metaaminophenylboronic acid. Can be obtained by [H. El Weiss,
Others, Biochemistry (HLWeith, et.al., Biochemis
try), 9 , 4396 ('70)].

It can also be obtained by reacting the hydroxyl group with bromocyan, followed by reaction with an oligopeptide and further reaction with metaaminophenylboronic acid in the presence of carbodiimide [USP 3,912,595].

Further, it can also be obtained by introducing epihalohydrin, bisepoxide or the like into the hydroxyl group by a known method, and then reacting with metaaminophenylboronic acid.

〔The invention's effect〕

The gel of the present invention is rigid. Therefore, it is unlikely to be damaged during handling. Also, when the column is packed and used for separation and analysis for liquid chromatography, the eluent can be flowed at a high flow rate, which enables rapid analysis. Further, since it can be used by making it into fine particles, high separation is expected.

Further, it is chemically stable and can be stably used even in an alkaline solution in which silica, glass and the like are easily dissolved. When a polymer having a dihydroxyboronyl group is used to analyze a compound having a 1,2 cis diol, it is preferably carried out on the alkaline side, and therefore it is important that the polymer is stable under alkaline conditions. Further, the gel of the present invention has a hydroxyl group. The styrene-divinylbenzene copolymer is poor in hydrophilicity and may exhibit hydrophobic adsorption for proteins and the like, but the gel of the present invention has a hydroxyl group and thus has high hydrophilicity and is suitable for proteins and the like. In contrast, it does not exhibit hydrophobic adsorption, which is preferable.

Example 1 Utility Model 57-30945 described in Japanese Example No.1 vinyl alcohol polymer gel (X; 0.3, the amount of hydroxyl _{groups; 7.8meq / g, W R;} 1.7
8g / g, specific surface area; 78m ² / g, particle size; 12.1μm) 25g, epichlorohydrin 116.8g, dimethyl sulfoxide 250m
l, add 12.5 ml of 10N sodium hydroxide aqueous solution, and add 20
Reacted for hours. The introduction amount of the epoxy group was 1.0 meq / g. The epoxy group was quantified by the method described in JP-A-57-190003.

Next, 13 g of aminophenylboronic acid hemisulfate and 250 ml of water were dissolved and 20 g of the above epoxy group-introduced polymer was added to pH 11 and reacted at 60 ° C. for 20 hours. Next, 25 mM tris (hydroxymethyl) aminomethane was added to protect the residual epoxy group, and the mixture was reacted at 50 ° C for 5 hours.

The amount of boric acid introduced was 0.28 meq / g. The quantification of boric acid was performed by the following method. The boron-carbon bond was cleaved by adding 10 ml of 30% hydrogen peroxide solution to 1 g of the above-mentioned polymer into which aminophenylboronic acid was introduced and reacting for 5 hours. Next, the polymer was spun down and the supernatant was collected.

After decarboxylation, boric acid ester was formed by adding sugar, strongly oxidized, and boric acid was quantified by titration with sodium hydroxide.

The instruments used for this analysis are all made of quartz.

The hydroxyl group density of the copolymer introduced with aminophenylboronic acid was 7.6 meq / g, and W _R was 1.70 g / g. The particle size and specific surface area were the same as before introduction.

The above aminophenylboronic acid-introduced copolymer has an inner diameter of 7.
It was packed in a stainless steel column having a length of 6 mm and a length of 100 mm to obtain a packed column for high performance liquid chromatography.

Application Example Using a column packed with an aminophenylboronic acid-introduced copolymer prepared by the method shown in the examples, glucose and fructose were separated by liquid chromatography under the following conditions. As shown in the figure, D glucose and D fructose could be separated.

Sample: 0.4% D-glucose + 1% D-fructose (20
μ) Mobile phase: 50 mM ammonium chloride (pH 5.5) Flow rate: 0.5 ml / min Pump; JASCO Corporation, trade name TRI ROTAR-V Detector; Showa Denko, trade name Shodex RI SE-51 Temperature; 25 ° C. example described in Japanese example No.2 2 Sho 57-30945 vinyl alcohol copolymer gel (X; 0.4, the amount of hydroxyl groups; 4.2meq / g, W _R;
An epoxy group-introduced copolymer was obtained by the same method as in Example 1, except that 2.48 g / g, specific surface area; 120 m ² / g, particle size; 9.6 μm) was used. The amount of epoxy groups was 0.6 meq / g. Then, using 20 g of the epoxy group-containing copolymer, a boric acid-introduced copolymer was obtained in the same manner as in Example 1. The amount of boric acid introduced is 0.
It was 2 meq / g. The hydroxyl group density of the aminophenylboronic acid-introduced copolymer was 4.0 meq / g, and W _R was 2.40 g / g. The specific surface area and particle size were the same as before the introduction.

[Brief description of drawings]

FIG. 1 is a chromatogram obtained in Example 1, where peak A is D-glucose and peak B is D-fructose.

Claims (1)

[Claims] 1. An alcoholic hydroxyl group derived from a vinyl alcohol unit based on the weight of the cross-linked copolymer, 1.0 to 14.0 meq / g, a dihydroxyboronyl group, 0.05 to 5.0 meq / g, and a specific surface area of 5 to 10.
00m ² / g, the amount of water that can be retained is 0.5 to 6.0 g / g, and the proportion of the crosslinkable monomer units in all the monomer units forming the gel is such that X in the formula (2) is 0.2X ≦ Boron-containing cross-linked copolymer chromatography gel in the range of 0.4. (However, a is a mole fraction of the monomer unit excluding the crosslinkable monomer unit in the copolymer, b is a mole fraction of the crosslinkable monomer unit, and n is one molecule of the crosslinkable monomer. Represents the number of functional groups such as a polymerizable vinyl group.)