JPH0352648A - Production of anion exchange resin - Google Patents

Abstract

PURPOSE:To obtain an anion exchange resin enabling quantitative analysis of anions even in a sample contg. protein by coating the surface of the base of an anion exchange resin with a synthetic polymer having negative charges. CONSTITUTION:Gel of a polymer such as a cross-linked hydroxyalkyl methacrylate polymer having functional groups such as epoxy groups is prepd. as the base of an anion exchange resin and dispersed in a buffer soln. in which a synthetic polymer having negative charges such as a 2-hydroxymethacrylate- methacrylic acid copolymer is easily fixed. The buffer soln. is further mixed with an aq. soln. of zinc borofluoride, etc., and the synthetic polymer having negative charges. The polymer is fixed on the gel by a reaction and the gel is washed and mixed with a compd. rendering an ion exchange group. This mixture is brought into a reaction and washed with water and exchange for desired counter ions is performed. The resulting anion exchange resin enables quantitative analysis of anions even in a sample contg. protein.

Description

Detailed Description of the Invention Industrial Field of Application The present invention is a liquid chromatography method (
The present invention relates to a method for producing an anion exchange resin (filler) suitable for quantitative analysis using ion chromatography (including ion chromatography).

Conventional Techniques In general, when anions in samples containing proteins are quantitatively analyzed using liquid chromatography (including ion chromatography), the following problems occur. In other words, proteins in the sample are adsorbed to the surface or ion exchange groups of the anion exchange resin packed in the separation column, reducing the apparent ion exchange capacity and decreasing the anion separation performance or reducing quantitative performance. was often decreased. In addition, the accumulation of adsorbed proteins in this manner often increases the pressure in the separation column, and when such a pressure increase occurs, it has become practically impossible to regenerate the separation column. Methods to solve these problems include removing proteins from the sample in advance, washing the separation column frequently to remove adsorbed proteins, and separating the switching valve equipped with a Brecolumn that adsorbs proteins. Methods such as installing it in front of a column to remove proteins were used. <Problems to be solved by the invention> In the case of a method in which proteins are removed from a sample in advance, firstly, it takes time to pre-process the sample;
Second, deproteinization agents may have a negative effect on the separation of target ions, and third, target components may be incorporated into the proteins that are removed by precipitation. ．． In addition, in the case of a method in which the separation column is frequently washed to remove adsorbed proteins, the analysis must first be stopped and a solvent different from the mobile phase flowed through the column, thus maintaining the continuity of the analysis. The second point is that it is not desirable from the viewpoint of ensuring maintenance efficiency.
The disadvantage was that once proteins were adsorbed to the column, they could not be easily washed away, resulting in rapid column wear. The method of removing proteins by installing a pre-column that adsorbs proteins in front of the separation column does not have the drawbacks mentioned above and is currently the best method, but the equipment is complex and maintenance of the pre-column (adsorption column) is required. The present invention was made in view of this situation, and its purpose is to remove anions from a sample packed in a chromatography tube and containing proteins using liquid chromatography. Means for solving problems in providing a method for producing an anion exchange resin (filling agent) suitable for quantitative analysis using one method (including one method of ion chromatography) 〉 The feature of the present invention that solves the above-mentioned problems is that in the method of manufacturing anion exchange resin (filling agent), it is packed into a chromato tube and used as a separation column for ion chromatograph or liquid chromatograph. The purpose is to produce an anion exchange resin using the following steps (a) to (e). (b) A process of preparing a polymer gel with a certain functional group according to the reaction method A process of preparing a resin with a certain functional group introduced according to the reaction method. (b) A step of dispersing the polymer gel in a buffer II in which negatively charged synthetic polymer substances tend to be immobilized. (c) A synthetic polymer substance having a negative charge to be bound is mixed and immobilized in the dispersion solution, and after the reaction is completed, the step of washing thoroughly with liquid j. (2) A process of mixing compounds that will become ion exchange groups and reacting them at a constant temperature for a certain period of time. (e) A step of washing the resin produced by the reaction with a buffer solution, thoroughly washing it with water, and then exchanging it with the desired counterion. <Example> The present invention will be explained in detail below using figures. 1st
The figure is a process diagram for explaining the method for producing an anion exchange resin (filler) according to the present invention. In this figure, first, a polymer gel with certain functional groups is prepared depending on the reaction method, or a resin with certain functional groups introduced is made depending on the reaction method. This is the base material.
An example is a hydroxyalkyl methacrylate-based crosslinked polymer gel with a particle size of 12 μm into which 350 μmol/g of epoxy groups have been introduced. Next, the above-mentioned polymer gel is dispersed in a solution in which a synthetic polymer substance with a negative charge is likely to be immobilized [#. To illustrate, 5 g of the above-mentioned do-O-oxyalkyl methacrylate-based crosslinked polymer gel
(dry weight), 1% 2-hydroxymethacrylate methacrylic acid (methacrylic acid content; weight ratio 20%)
It was dispersed in a cobolymer aqueous solution, 0.05 g of a 45% zinc borofluoride aqueous solution was added, and the mixture was reacted in an incubator at 50'C for 6 hours. After the reaction, the resin is thoroughly washed with pure water, a compound that will become an ion exchange group is mixed, and the mixture is allowed to react at a constant temperature for a certain period of time. To give an example, 10
% trimethylamine solution and incubated at 30°C.
Allow to react for 0 hours, and after the reaction is complete, wash the resin thoroughly with pure water. After that, O. Dispersed in IM sulfuric acid aqueous solution,
Incubate at °C for 2 hours. Next, the resin produced by the reaction is washed with M solution and then thoroughly with water, and then exchanged with the desired counterion. For example, the resin produced by the reaction is thoroughly washed with Looml pure water, then dispersed and filtered in a 0.5M aqueous sodium chloride solution, and further washed with 50ml of a 0.5M aqueous sodium chloride solution. After that, it was thoroughly washed with pure water, dispersed in a 0.1M sodium chloride aqueous solution, and
Leave it overnight. The anion exchange resin obtained as illustrated had an ion exchange capacity of 45 μEq/ml. Moreover, this anion exchange resin has an inner diameter of, for example, 5.
It is used as a separation column for ion chromatographs and liquid chromatographs by filling stainless steel chromato tubes with a diameter of 0 mm and a length of 100 mm using the high-pressure slurry filling method. By the way, the above-mentioned base material is either a relatively hydrophilic porous cross-linked polymer gel with a functional group on its surface that can supply a synthetic polymer substance with a negative charge, or a material with a negative charge on its surface. The resin must be able to introduce functional groups to which synthetic polymeric substances can be bonded. ．． Additionally, the negatively charged synthetic polymer substance may have a reactive functional group such as an epoxy group that forms a bond with the base material. Furthermore, the pores of such resins must be so small that negatively charged synthetic polymeric substances bound to the surface or proteins present in the sample cannot penetrate, or only a small amount can penetrate. in particular,
Resins such as (a) or (b) below are desirable. (a) Polymethacrylate resins, polyvinyl alcohol resins, polyether resins, etc. having carboxyl groups, amine groups, hydroxyl groups, or epoxy groups. (b) A polymethacrylate resin having a halogen group or a hydroxyl group and into which a functional group to which the above-mentioned functional group and a negatively charged synthetic polymer substance can be chemically bonded can be introduced. Polyvinyl alcohol resin, polyedel resin, etc. Furthermore, the negatively charged synthetic polymer substance must have a molecular weight that allows it to react with the functional groups of the base material and prevent it from penetrating into the pores of the base material. A negatively charged polymeric substance has sufficient hydrophilic groups (e.g., hydroxyl group) that are uncharged in the mobile phase used in the polymer chain, and at the same time contains negatively charged groups (e.g., carboxyl group). , sulfone group, etc.). At this time, the mole fraction of the hydrophilic group and the negatively charged group in the polymer chain is 0.05 to 0.05 for the negatively charged group.
0.5, preferably 0.1-0.3. Examples of such synthetic polymers include 2-hydroxyethyl methacrylate-methacrylic acid copolymer. In this case, the hydroxyl group of 2-hydroqethyl methacrylate is a hydrophilic group and at the same time is a functional group that reacts with the base material. Furthermore, instead of a hydrophilic group, the material may have a functional group (for example, an epoxy group) that can be converted into a hydrophilic group by chemical treatment after bonding a polymer to the surface of the substrate. At this time, the molar fraction of the hydrophilic group and the negatively charged group in the polymer chain must be 0.05 to 0.7, preferably 0.1 to 0.5 for the negatively charged group. Must be. As an example of such a synthetic polymer, 2
-Hydroxyethyl methacrylate-methacrylic acid copolymer and the like.

The ion exchange group inside the pores of the base material is a low-molecular material that can sufficiently penetrate into the inside of the pores of the base material. It must be a substance that reacts with the functional group (which does not have to be the same as the functional group to which the synthetic polymer substance is bonded) to form an anion exchange group. Specifically, this includes common alkylamines, alkanolamines such as ethanolamine, diamines, and hydroxyalkylammonium. FIG. 2 is an explanatory diagram of the structure of a general sub-rest type ion chromatography device. When the liquid supply bomb 2a is driven, the eluent in the eluent tank 1a is transferred from the liquid supply bomb 2a to the injector 3 to the separation column 4. →Flows through the inner chamber 5c of the suppressor 5 and the detector 6 to the waste liquid tank 7a. Furthermore, when the liquid sending bomb 2b is driven, the removal liquid in the removing liquid tank 1b passes from the liquid sending bomb 2b to the outer chamber 5b of the sublessor 5, and then passes through the waste liquid tank 7.
Flows to b. Therefore, the cations existing in the inner chamber 5c of the suppressor 5 are ion-exchanged with the outer chamber 5b of the suppressor 5 through the cation exchange 11i5a, and as a result, the fluid present in the inner chamber 5c of the suppressor 5 is The conductivity background is removed. The separation column 4, suppressor 5, and detector 6 are housed in a constant temperature bath 9 and kept at a constant temperature (for example, 40'C), and the liquid supply pump 2. Injector 3, separation column 4, suppressor 5.
The detector 6 is often housed within the casing 10 of the analyzer. In an ion chromatograph apparatus having such a configuration, anions contained in a sample injected in a fixed amount into the injector 3 are separated in the separation column 4, and then the conductivity is reduced in the sublessor 5 as described above. After the background is removed, it is detected by detector 6. The signals detected by the detector 6 in this manner are guided to a display device 8 (for example, a recorder) to draw a chromatogram. The anion exchange resin produced as described above was packed into separation column 4, and the samples described below were collected and analyzed under the following experimental conditions, and the following experimental results were obtained. That is, (a) anion exchange resin was filled into a stainless steel chromatography tube with an inner diameter of 5.0 mm and a length of 100 mm using a high-pressure slurry filling method, and the recovery rate of the protein chamber was measured in a phosphoric acid solution. , about 95% (albumin).
O3 /4.0mM NaHCO:+ (pH is 10.0
), the recovery rate was approximately 88%. (D) 4.0mM Na2 CO3 /4. OmMNaH
A mobile phase of CO3 (PH is 10.0) was used (flow rate was 2.0%).
0ml/min. )L, temperature of constant temperature bath 9 is 40°C. Sample injection bag 50μl. The type of detector 6 was an ultraviolet absorption detector, and CI- ions, N O 2 ions, BY'''' ions, N O 3- ions, and SO a2- ions were successfully separated. (c) A sample containing 0.2% albumin was measured under the same conditions as in <b) above, but in this case as well, CI- ions, NO2- ions, BY- ions, N03- ions, SO42 -Ions could be separated well. From the above, it can be seen that by using the anion exchange resin of the present invention, low-molecular-weight anions in samples containing proteins can be analyzed without the influence of proteins. By the way, the anion exchange resin obtained by the production method according to the present invention has a structure as shown in Figure 3. That is, a large number of polymers (specifically, carboxyl groups, etc.) B, which dissociate and have a weak negative charge when the pH value is about 10, are bonded to the surface of the anion exchange resin A. In addition, on a part of the surface of the anion exchange resin A, there is a pore D modeled as a wedge shape (actually it is said to be shaped like a complicated cave). A large number of anion exchange groups C are bonded. In an anion exchange resin with such a structure, the protein P in the sample has a large molecular radius, so the pore D
Moreover, since it has a negative charge, the ion is excluded by the polymer B. Further, the anion S1 in the sample undergoes anion exchange with the anion exchange group C in the pore D, and the cation S+ in the sample undergoes extremely weak cation exchange with the polymer B. On the other hand, when ion exchange chromatography is performed using the anion exchange resin obtained by the production method according to the present invention, the behavior in which sample components are retained in the packing material is considered to be as follows. That is, considering the analysis in am liquid with a pH value of 10, at a pH value of 10, the carboxylic groups are dissociated and have a negative charge, and the quaternary ammonium groups present inside the filled pores are also dissociated. It has a positive charge. Under these conditions, for example, the retention behavior when a sample containing a protein with an isoelectric point of 5 is injected is as follows. That is, if the ion exchange group inside the filler pores is of the quaternary ammonium type, 11! r
It dissociates even in liquid and takes on a positive charge. When a low-molecular anion approaches such a filler, the ion is removed by the negative charge of the carboxyl group in the polymer (synthetic polymer material) on the surface of the anion exchange resin, but the anion inside the pores is removed. Because the exchange group has a stronger positive charge, low-molecular anions enter the pores and are adsorbed through ion exchange. Furthermore, when a protein whose isoelectric point is smaller than that of the buffer solution approaches this packing material, the sample protein is also negatively charged, so it is subjected to ion exclusion by the negative charge on the surface of the anion exchange resin. It is possible that proteins are attracted by the positive charge of the anion exchange groups inside the pores, but proteins have a large molecular weight and cannot penetrate into the pores, so they cannot be ion-exchanged and adsorbed with the ion exchange groups inside the pores, and elute. It will be done.

In fact, due to some hydrophobic adsorption, some is retained in the filler. In the case of low-molecular cations, they are adsorbed by ion exchange to the negative charges of the carboxyl groups in the polymer on the resin surface, but since the negative charges on the surface are extremely small, they can be easily desorbed from the resin even with a mobile phase of low concentration. Put it away. When proteins and low-molecular-weight anions coexist in a sample, the above two phenomena occur simultaneously, and only the low-molecular-weight anions are retained by the packing material and separated.Summary of the above phenomena. and becomes as follows. That is. (stomach)
When proteins and enzymes coexist, ion exclusion occurs between the negative charges of the carboxyl groups in the polymer on the resin surface. In addition, proteins and enzymes cannot penetrate into the pores of the filler because they are large in size. For this reason,
Coupled with the fact that the amount of negative charge on the surface is extremely small, even a mobile phase with a low concentration can be easily desorbed from the resin.
Holding power is small. c) In the case of low-molecular anions, the ions are excluded between them and the negative charge of the carboxyl group in the polymer on the resin surface.
It can also penetrate into the pores of the filler and exchange ions with anion exchange groups inside the pores. Therefore, the holding force is large. (c) In the case of low-molecular-weight cations, they are ion-exchanged and adsorbed to the negative charges of carboxyl groups in the polymer on the resin surface. It can also penetrate into the pores of the filler, but ions are excluded inside the pores. Therefore, since the amount of negative charge on the surface is extremely small, even a mobile phase with a low concentration can be easily desorbed from the resin. Therefore, the holding force is small. Effects of the Invention> According to the practical examples of the present invention as explained in detail above, anions in a sample packed in a chromatography tube and in which proteins coexist can be detected using a liquid chromatography method (including an ion chromatography method). A method for producing an anion exchange resin (filling agent) suitable for quantitative analysis has been realized. In addition, the anion exchange resin produced in this way has the surface of the base material coated with a synthetic polymer that has a negative charge, so that low-molecular anions in samples containing proteins can be absorbed without being affected by proteins. It has the advantage of being measurable. Furthermore, since samples containing proteins etc. can be directly injected without the need for extra operations such as washing, analysis conditions are simple and measurement time is significantly reduced.
In addition, the problem of shortening retention time due to an apparent decrease in ion exchange capacity caused by adsorption of proteins in the sample to the column packing material is eliminated, and as a result, a chromatogram with good reproducibility can be obtained. There is also the advantage of being able to Another advantage is that column life can be extended by eliminating deterioration in column performance caused by increases in column pressure due to adsorption of proteins in the sample to the column packing material.

[Brief explanation of drawings]

Figure 1 is a process diagram for explaining the method for producing anion exchange resin (filler) according to the present invention, Figure 2 is a schematic diagram of a general ion chromatography device, and Figure 3 is 1 is a horizontal view of an anion exchange resin obtained by the manufacturing method according to the present invention. 1a... Eluent tank, 2a, 2b.
...Liquid pump, 3...Injector,
4... Separation column, 5... Sagressor, 6... Detector, 7... Waste liquid tank, 8...
... Display device, 9 ... Constant temperature chamber, 10 ...
... Analyzer housing diagram

Claims (4)

[Claims] (1) Anion exchange resins packed in chromatography tubes and used as separation columns for ion chromatography are listed below (
A method for producing an anion exchange resin, characterized in that it is produced by the steps of (a) to (e). (a) A process of preparing a polymer gel with a certain functional group according to the reaction method A process of preparing a resin with a certain functional group introduced according to the reaction method. (b) A step of dispersing the polymer gel in a buffer solution in which negatively charged synthetic polymer substances are likely to be immobilized. (c) A step of mixing and immobilizing a synthetic polymer substance with a negative charge to be bound into the dispersion solution, and washing thoroughly with the buffer solution after the reaction is completed. (d) A step of mixing compounds that will become ion exchange groups and reacting them at a constant temperature for a certain period of time. (e) A step of thoroughly washing the resin produced by the reaction with water and then exchanging it with the desired counterion. (2) The base material is a relatively hydrophilic crosslinked polymer gel made of a resin having a functional group on its surface to which a synthetic polymer substance having a negative charge can be bonded. 1) A method for producing an anion exchange resin as described in section 1). (3) The negatively charged synthetic polymer substance has a molecular radius that reacts with the functional group of the base material and cannot penetrate into the pores of the base material, and has a molecular radius that is used in the polymer chain. Production of an anion exchange resin according to claim (1), which has a sufficient number of uncharged hydrophilic groups in the phase and partially has functional groups that exhibit a negative charge. Method. (4) The ion exchange group can sufficiently penetrate inside the pores of the base material and react with unreacted functional groups remaining inside the pores without bonding with the negatively charged synthetic polymer substance. The method for producing an anion exchange resin according to claim (1), wherein the substance is a substance that serves as an anion exchange group.