JPH03146507A - Production of boric acid type resin - Google Patents

Abstract

PURPOSE:To obtain the subject resin without increasing column pressure and deteriorating column performance by slurrying a substrate polymer gel having pores with a small inside diameter in a solution, forming a hydrophilic polymer layer on the surface thereof, washing the resultant reaction product and reacting a compound capable of providing boric acid group therewith. CONSTITUTION:A substrate polymer gel having pores with a small inside diameter and prescribed functional groups is prepared and subsequently dispersed in a solution capable of readily forming a surface polymer layer to provided a slurry. A hydrophilic polymer or a compound to be bound thereto is then mixed and reacted therewith, as necessary, simultaneously crosslinked and the resultant reaction product is subsequently washed with pure water and an alcohol to provide a resin having a polymer layer with high hydrophilicity on the surface thereof. The resultant layer is then mixed with a compound to provide boric acid group and reacted therewith at a prescribed temperature for a prescribed time to introduce the boric acid groups into the resin. The resin obtained by the reaction is subsequently washed with a buffer solution and then sufficiently washed with water to afford the objective boric acid type resin.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for producing a boric acid type resin, and more specifically, to a method for producing a boric acid type resin, and more specifically, a method for converting a substance having cis-glycol type and catechol type complements in a biological sample into a material such as a protein. This invention relates to a method for producing a resin having a boric acid type functional group that can be adsorbed onto cotton without the influence of oxidation.

<Prior Art> As an adsorbent for cis-glycol type and catechol type substances in biological samples (serum, plasma, urine, etc.), a resin in which a boric acid functional group is introduced into acarose is known. This resin is made by introducing 20-40 μm o I/g of boric acid into an agarose gel with a particle size of 0.1-0.4 mm via a spacer, and is mainly used to selectively target glycoproteins. It was created for supplementary purposes. but,
Since it exhibits specific adsorption ability for catechol-type substances, it can also be used as an adsorption resin for catecholamines. Several pretreatment methods and analysis methods using this boric acid-introduced agarose gel have already been reported, but they have the following problems in practical terms. First, agarose gel is made by cross-linking agarose with epichlorohydrin or the like, and because it exhibits good hydrophilicity, it is used as a carrier for gel filtration chromatography or affinity chromatography of proteins.

However, since it is very soft, it is not possible to improve its performance by making it into fine particles, so the particle size that can be used becomes large, and as a result, the dependence of the adsorption characteristics on the flow rate becomes strong. Furthermore, due to the softness of the base material, it is difficult to use in high performance liquid chromatography and lacks long-term stability.

Second, regarding the introduction of boric acid, a spacer is used to facilitate the adsorption of glycoproteins, but spacers are not necessarily effective for adsorbing only low-molecular substances other than glycoproteins. In other words, when trying to adsorb catechol-type low molecular weight substances such as catecholamines in a sample, selective adsorption cannot be performed because sugars and glycoproteins coexisting in the sample are also adsorbed, and the excess The adsorption amount of catecholamines may decrease due to the adsorption of components.

Thirdly, regarding the amount of boric acid introduced, it is 20 to 40 μm o
l/g, but since a resin introduced in a large amount can obtain higher adsorption power with a smaller amount of resin, the amount introduced needs to be larger.

<Problems to be Solved by the Invention> The present invention has been made in view of the above situation, and its purpose is to remove substances with cis-glycol type and catechol type structures from biological samples without being affected by proteins, etc. An object of the present invention is to provide a method for producing a resin having a boric acid type functional group that can be absorbed and concentrated.

Specific Means for Solving the Problems> The present invention provides a method for producing a boric acid resin, in which the boric acid resin is
It is characterized by being manufactured by the process (2).

(2) A step of preparing a polymer gel with a predetermined functional group according to the reaction method, or creating a resin with a predetermined functional group introduced according to the reaction method.

(2) A step of dispersing the polymer gel in a solution in which a surface polymer layer is easily formed to form a slurry.

(2) A step of mixing and reacting a hydrophilic polymer or compound to be bonded with the slurry and, if necessary, post-crosslinking.

■After the reaction, the process is thoroughly washed with pure water and alcohol.

■A process in which compounds that will become boric acid groups are mixed and reacted at a constant temperature for a certain period of time.

■The step of washing the obtained resin with a buffer solution and then thoroughly washing it with water.

<Example> Hereinafter, the present invention will be explained in detail using the drawings. 1st
The figure is a flowchart for explaining the method for producing boric acid type resin according to the present invention. As shown in this figure, the boric acid type resin according to the present invention is manufactured by the following steps (1) to (6). That is, (1) prepare a polymer gel having a predetermined functional group according to the reaction method, or prepare a resin into which a predetermined functional group is introduced according to the reaction method;

(2) Disperse the above gel in a solution that facilitates the formation of a surface polymer layer to form a slurry.

(2) A polymer or compound to be bonded is mixed with the above slurry and reacted. Further, if necessary, post-crosslinking is performed.

■After the reaction, wash thoroughly with pure water and alcohol.

■Mix the compounds that will become boric acid groups and let them react at a constant temperature for a certain period of time.

(2) Wash the obtained resin with a buffer solution and then thoroughly with water.

The boric acid type resin thus obtained is filled into a suitable ROMAT tube and used as a pretreatment column for adsorption and concentration.

By the way, in the resin obtained as described above, the hydrophobicity of the base material due to the high degree of crosslinking to obtain hardness is covered by a hydrophilic polymer layer on the surface of the base material, making it extremely hydrophilic. It becomes an expensive resin. This hydrophilic polymer layer suppresses protein adsorption. In addition, since boric acid groups are bonded directly or with very short chains to the surface hydrophilic polymer layer, not only can the amount of boric acid introduced be larger than that of the spacer type, but polymers with large steric hindrance are Even if it has a functional group that can be adsorbed, it cannot be strongly adsorbed. Furthermore, because the pore diameter is extremely small, glycoproteins cannot penetrate into the pores, and they simply form a bond with the other boric acid groups left on the surface, which is almost always size-exclusive. It elutes into

On the other hand, the boric acid resin obtained as described above is produced by forming a highly hydrophilic polymer layer on the surface of the base polymer gel, and then reacting it with a compound having a boric acid group without using a spacer. It is a resin into which groups have been introduced.

Here, the base material is a relatively hydrophilic porous crosslinked polymer gel, and the degree of crosslinking is 20% by weight to maintain hardness.
The surface must have a functional group to which the polymer that will form the desired surface polymer layer can be bonded and formed, or the surface must have a functional group to which the desired polymer layer can be bonded and formed. The resin must be capable of introducing functional groups. In addition, the pores of the resin must be so small that they cannot be penetrated by the proteins present in the sample, or even a tiny bit. As a rough guide, the exclusion limit molecular weight is 10,000 or less, preferably 3.
The resin must have pores of about 0.000 to 8,000. As the base material, the following materials A and B can be used. That is, A: polymethacrylate, polyvinyl alcohol, polyether resin, etc., having a carboxyl group, amine group, formyl group, hydroxyl group, or epoxy group, etc. B: having a halogen group or a hydroxyl group, and the functional groups shown in A above and the following: Polymethacrylate, polyvinyl alcohol, polyether resin, etc., into which a functional group to which a target polymer can be chemically bonded can be introduced, and a highly hydrophilic polymer layer can be bonded or formed as follows.

■ A hydrophilic polymer that has a functional group that can react with the functional group of the above base material and that can react with a boric acid compound, or into which such a functional group can be introduced, is grouped in a solution. Make the material react.

■It is an organic compound that has a functional group that can react with the functional group of the above base material, has a functional group that can react with a boric acid compound, or has a functional group that can introduce such a functional group. The substrate is reacted with a substance that polymerizes during the reaction with the substrate or can be crosslinked after the reaction.

As examples of (2), polysaccharides such as cellulose, polyvinyl alcohol, etc. can be used, and as examples of (2), polyglycidyl ethers of polyols, polyhydric alcohols, etc. can be used.

A boric acid group is a compound that can introduce a boric acid group by reacting with a functional group of a surface-bound polymer or a functional group introduced into a surface-bound polymer. For example, the commonly used aminophenylboric acid is a typical example. Alkylboronic acids with amine groups can be used.

Next, an A#-like example (experimental example) conducted by the present inventors
I will explain about it. Initially, a copolymer gel of ethylene glycol dimethacrylate and glycidyl methacrylate (
Degree of crosslinking: 35% by weight, average particle size: 11 μm, exclusion limit molecule: FF 13000 [measured with sugars]> 5 g and sorbitol polyglycidyl needle (3 g) were mixed in 50 ml of dimethyl sulfoxide containing 50 μm of boron trifluoride etherate at 40°C for 2 hours. The reaction forms a hydrophilic polymer layer.

5 g (wet weight) of this hydrophilic coating gel was added to 30 ml of I
After dispersing in an aqueous N sodium hydroxide solution and adding 3 g of epichlorohydrin, the mixture was incubated in an incubator at 40'C for 2 hours. After the reaction, the resin was thoroughly washed with pure water, methanol, and pure water in this order.

The resin after the reaction was dispersed in a 1% aqueous solution of tetrabutylammonium hydroxide, and 0.5 g of aminophenylboric acid was added and dissolved therein, followed by reaction at 40'C for 4 hours.

After the reaction, the resin was washed with pure water and alcohol, then washed again with pure water and 0.1M sodium chloride, and then dispersed in pure water.
I left it for the night.

When the adsorption capacity of the boric acid type resin obtained as described above was measured using dopamine, it was found to be about 140 nmol/
It was possible to adsorb g of dopamine.

After that, the aforementioned boric acid gel was coated with an inner diameter of 4.6 mm and a length of 1 mm.
Packed into a 0 mm stainless steel column, 50 mM phosphate buffer (pH 8.5) was pumped to remove catecholamines 3.
A standard solution containing seeds (epinephrine, norepinephrine, dopamine) at 20 μs/mj each, serum, and serum to which the standard solution had been added were injected to examine the degree of adsorption of thecolaminos and other substances. The mobile phase flow rate is l
The injection rate was ml/min, and the sample injection amount was 20μ. A diode array type ultraviolet absorption detector was used as a detector, and the eluted components were monitored in the range of 200 to 400 nm. The column temperature was 40°C.

Catecholamines eluted as a very broad peak after 3 m1n. When serum and serum added with catecholamines were injected, the elution of a large peak started at about 0.15 ml, and at about 1.6 ml.
It returned to the baseline at n, and did not overlap with the peak of catecholamines at all.

Next, the recovery rates of albumin and lysozyme were determined using the same buffer solution.

Both albumin and lysozyme showed a good recovery rate of 98%, and it was found that there was no adsorption to the filler.

Furthermore, similar measurements were carried out by increasing the measurement flow rate to 2 ml/min, but there was no change in the adsorption capacity, indicating that it can be used at a high flow rate. After the measurement, the end of the column was opened to check the packing state, but no dents or the like were observed.

Effects> As described in detail above, according to the present invention, in the method for producing boric acid resin, the boric acid resin is produced in a predetermined process consisting of five steps. Therefore, the following effects can be obtained. That is, (1) the hydrophobicity of the base material can be suppressed by coating the base material surface with a highly hydrophilic polymer, and cis-glycol type and catechol type substances in samples containing proteins can be It has become possible to selectively adsorb and concentrate without any adverse effects.

■Since samples containing proteins, etc. can be directly injected without the need for extra operations such as washing, adsorption/desorption conditions are simple and measurement time is significantly reduced.

■This eliminates the problem of changes in the amount of supplementation caused by the apparent decrease in adsorption force caused by the adsorption of proteins and other substances in the sample onto the packing material, making it possible to adsorb and concentrate with good reproducibility.

■Since boric acid is introduced directly, the amount introduced is larger than with the spacer type, and it is possible to adsorb it sufficiently with a small amount of resin.

■Because it uses a hydrophilic polymer with a high degree of crosslinking, +18
It can be used at high flow rates even when using a bulk filler, and there are no problems with the flow rate dependence of the amount of adsorption or the lifetime.

The increase in column pressure and the deterioration of column performance due to adsorption of proteins and other substances in sample No. 1 to the packing material were eliminated, and the column life was extended.

[Brief explanation of the drawing]

The figure is a flowchart for explaining an embodiment of the present invention.

Claims (6)

[Claims] (1) After forming a highly hydrophilic polymer layer on the surface of a base polymer gel having pores with a small inner diameter, a resin containing boric acid groups is introduced by reacting with a compound having boric acid groups. A method for producing a boric acid type resin, characterized by producing it through the following steps (1) to (6). (1) A step of preparing a polymer gel having a predetermined functional group according to the reaction method, or producing a resin having a predetermined functional group introduced according to the reaction method. (2) A step of dispersing the polymer gel in a solution in which a surface polymer layer is easily formed to form a slurry. (3) A step of mixing and reacting a hydrophilic polymer or compound to be bonded with the slurry and, if necessary, post-crosslinking. (4) After the completion of the reaction, washing with pure water and alcohol. (5) A step of mixing compounds that will become boric acid groups and reacting them at a constant temperature for a certain period of time. (6) A step of washing the resin obtained in the reaction with a buffer solution and then thoroughly washing with water.