JP2830107B2 - Anion exchange resin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial application field> The present invention uses liquid chromatography (including ion chromatography) to detect anions in a sample packed in a chromatographic tube and coexisting with proteins. Anion exchange resin suitable for quantitative analysis (filler)
About.

<Conventional Technology> In general, the following problems occur when quantitatively analyzing anions in a sample in which proteins coexist using liquid chromatography (including ion chromatography). In other words, proteins and the like in the sample are adsorbed on the surface of the anion exchange resin or ion exchange groups packed in the separation column, and the apparent ion exchange capacity is reduced, resulting in reduced anion separation performance or quantitative performance. Often decreased. Further, the pressure of the separation column often rises due to the accumulation of the protein adsorbed in this manner, and if such a pressure rise occurs, the regeneration of the separation column has become practically impossible. Methods for solving such problems include a method of removing proteins from a sample in advance, a method of frequently washing the separation column to remove adsorbed proteins, and a method of separating a switching valve provided with a precolumn for adsorbing proteins. A method of removing a protein by setting it in front of a column has been performed.

<Problems to be Solved by the Invention> However, in the case of a method for removing proteins from a sample in advance, firstly, pretreatment of the sample takes time, and the second deproteinizing agent has an adverse effect on the separation of ions to be measured. Third, there is a drawback that the target component is incorporated into the protein removed by precipitation or the like. In the case of a method for removing the adsorbed protein by frequently washing the separation column, the first method is used.
The analysis must be temporarily stopped and a solvent different from the mobile phase must be flowed through the column, which is not preferable from the viewpoint of maintaining continuity of analysis or securing maintenance efficiency, etc.
Second, once adsorbed proteins cannot be easily washed away, the column is quickly consumed. The method of removing proteins by placing a pre-column for protein adsorption in front of the separation column is currently the best method without the above-mentioned drawbacks, but the equipment is complicated and maintenance of the pre-column (adsorption column) is required. However, there is a new drawback in that

The present invention has been made in view of such circumstances,
The purpose is to use an anion exchange resin suitable for quantitative analysis using liquid chromatography (including ion chromatography) of anions in a sample packed in a chromatographic tube and coexisting with proteins. To provide.

<Means for Solving the Problems> In order to achieve such an object, the present invention provides a spherical anion exchange resin for separating and quantifying anions in an aqueous solution in which a protein coexists. Spheroids are attached to the surface and have a negatively charged polymer that repels the charge charged by the protein; micropores throughout the structure that are smaller than the molecular diameter of the protein; An anion exchange group bound to the inner surface of the micropore and ion-exchanging the anion entering the micropore, comprising: a quantitative analysis of the anion in a pH region where the protein has a negative charge. It is characterized by doing.

<Example> Hereinafter, the present invention will be described in detail with reference to the drawings.

FIG. 1 is a configuration diagram of the anion exchange resin of the present invention, which is partially enlarged.

The anion exchange resin is in the form of a sphere, and its surface has a weak negative charge when the pH value is about 10.
(For example, a carboxyl group) are bonded in large numbers.
In addition, a part of the surface of the anion exchange resin A has fine pores (a part shown in a wedge shape in the figure, hereinafter referred to as pores D). Are combined. In the anion exchange resin having such a structure, the protein P in the sample cannot enter the pores D due to a large molecular radius, and has a negative charge, so that it is ion-excluded by the polymer B. Further, the anion S in the sample ^- is an anion-exchange groups C and anion exchange in the pores D, ⁺ cations S in the sample are adapted to very low cation exchange with the polymer B.

FIG. 2 is a process explanatory view for explaining a method for producing an anion exchange resin according to the present invention. In this figure,
First, a polymer gel having a certain functional group is prepared according to the reaction method, or a resin having a certain functional group introduced therein is prepared according to the reaction method. This is the substrate. For example, 350 μmol / g epoxy group-introduced particle size
12 μm hydroxyalkyl methacrylate-based crosslinked polymer gel.

Next, the polymer gel is dispersed in a buffer in which a negatively charged synthetic polymer substance is easily immobilized. For example, 5 g (dry weight) of the hydroxyalkyl methacrylate-based crosslinked polymer gel is dispersed in a 1% aqueous solution of 2-hydroxymethacrylate / methacrylic acid (methacrylic acid content; 20% by weight) copolymer, 0.05 g of a 45% aqueous zinc borofluoride solution was added, and the mixture was reacted in an incubator at 50 ° C. for 6 hours. After the reaction, the resin is sufficiently washed with pure water, and then a compound to be an ion exchange group is mixed and reacted at a certain temperature for a certain time. For example, 10%
In a trimethylamine solution, and reacted at 30 ° C. for 10 hours. After the reaction, the resin was sufficiently washed with pure water.

Then, it is dispersed in a 0.1 M aqueous sulfuric acid solution and reacted at 30 ° C. for 2 hours.

Next, the resin generated by the reaction is washed with a buffer solution, and then sufficiently washed with water, and then exchanged with a target counter ion.
For example, the resin generated by the reaction is sufficiently washed with 100 ml of pure water, then dispersed and passed through a 0.5 M aqueous sodium chloride solution, and further washed with 50 ml of a 0.5 M aqueous sodium chloride solution. Wash thoroughly with pure water, disperse in 0.1M aqueous sodium chloride solution and leave overnight. The anion exchange resin obtained as illustrated had an ion exchange capacity of 45 μEq / ml. The anion exchange resin is filled into a stainless steel chromatograph tube having an inner diameter of 5.0 mm and a length of 100 mm using a high-pressure slurry filling method, and is used as a separation column for an ion chromatograph or a liquid chromatograph.

By the way, the base material is a relatively hydrophilic porous cross-linked polymer gel and has a functional group capable of supplying a synthetic polymer substance having a negative charge on its surface, or has a negative charge on its surface. The resin must be capable of introducing a functional group capable of binding to the synthetic polymer substance. Further, the synthetic polymer substance having a negative charge may have a reactive functional group that forms a bond with the substrate, such as an epoxy group. Further, the pores of such a resin must be so small that a synthetic polymer substance having a negative charge and a protein present in a sample, which are bound to the surface, cannot penetrate or can penetrate only slightly. Specifically, the following resin (a) or (b) is desirable.

(A) Polymethacrylate resin having a carboxyl group, amino group, hydroxyl group or epoxy group, polyvinyl alcohol resin, polyether resin, or the like.

(B) A polymethacrylate resin, a polyvinyl alcohol resin, or a polyether resin having a halogen group or a hydroxyl group, and capable of introducing a functional group capable of chemically bonding to the above-mentioned functional group and a synthetic polymer having a negative charge. Such.

Further, the synthetic polymer substance having a negative charge must have a molecular weight that reacts with the functional group of the substrate and cannot penetrate into the pores of the substrate. Synthetic polymer substances having a negative charge include groups having a sufficient amount of a hydrophilic group (eg, a hydroxyl group) having no charge in the mobile phase used in the polymer chain and a group having a negative charge at the same time (eg, a carboxyl group, (E.g., a sulfone group). At this time, the molar ratio of the hydrophilic group and the group having a negative charge in the polymer chain is 0.05 to 0 for the group having a negative charge.
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-hydroxyethyl methacrylate is a hydrophilic group, and at the same time, a functional group that reacts with the base material. Further, instead of the hydrophilic group, a functional group (for example, an epoxy group or the like) that can be exchanged for a hydrophilic group by a chemical treatment after bonding a synthetic polymer to the substrate surface may be used.
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.
Must be 0.7, preferably 0.1-0.5. Examples of such synthetic polymers include 2-hydroxyethyl methacrylate-methacrylic acid copolymer.

The ion exchange group inside the pores of the substrate is a low molecular weight material that can penetrate sufficiently into the pores of the substrate. (It does not have to be the same as the functional group to which the synthetic polymer substance is bonded.) Specific examples include general alkylamines, alkanolamines such as ethanolamine, azines, and hydroxyalkylammonium.

FIG. 3 is a configuration explanatory view of a general suppressed type ion chromatograph apparatus, and when the liquid feed pump 2a is driven,
The eluent in the eluent tank 1a flows to the waste liquid tank 7a via the liquid feed pump 2a → the injector 3 → the separation column 4 → the inner chamber 5c of the suppressor 5 → the detector 6. When the liquid feed pump 2b is driven, the removal liquid in the removal liquid tank 1b flows to the waste liquid tank 7b via the liquid supply pump 2b → the outer chamber 5b of the suppressor 5. For this reason, the cations existing in the inner chamber 5c of the suppressor 5 ion-exchange with the outer chamber 5b of the suppressor 5 via the cation exchange membrane 5a, and as a result, the fluid existing in the inner chamber 5c of the suppressor 5 Is removed.
The separation column 4, the suppressor 5, and the detector 6 are housed in a thermostat 9 to be maintained at a constant temperature (for example, 40 ° C.), and the liquid feed pump 2, the injector 3, the separation column 4, and the suppressor 5 , And the detector 6 are often housed in the housing 10 of the analyzer. In the ion chromatograph having such a configuration, anions contained in a sample injected into the injector 3 in a fixed amount are separated by the separation column 4, and thereafter, the conductivity of the anion is reduced by the suppressor 5 as described above. After the background is removed, it is detected by the detector 6. The signal detected by the detector 6 in this manner is
The display device 8 (for example, a recorder) draws a chromatogram.

The separation column 4 was filled with the anion exchange resin produced as described above, and a sample described later was collected and analyzed under the following experimental conditions. The following experimental results were obtained. That is, (a) Anion exchange resin was filled into a stainless steel chromatograph tube having 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 phosphate buffer. It was about 95% (albumin). Also, 4.0mMN
The recovery with a ₂ CO ₃ /4.0 mM NaHCO ₃ (pH 10.0) was about 88%.

(B) A mobile phase of 4.0 mM Na ₂ CO ₃ /4.0 mM NaHCO ₃ (pH 10.0) was used (flow rate was 2.0 ml / min.).
The sample injection amount is 50 μ, the type of the detector 6 is an ultraviolet absorption detector, and Cl ⁻ ions, NO ₂ ⁻ ions, Br ⁻ ions, NO ₃ ⁻ ions,
SO ₄ ^2- ion was successfully separated.

(C) A sample containing 0.2% of albumin was measured under the same conditions as in (b) above. In this case, too, Cl ^- ion, NO ₂ ^- ion, Br ^- ion, NO ₃ ^- ion, SO _{3- 4} ^2- Ions were successfully separated.

From the above, it can be seen that the use of the anion exchange resin of the present invention enables analysis of low molecular anions in a sample containing a protein without the influence of the protein.

It is considered that the behavior in which the sample components are retained in the filler is as follows. That is, considering the analysis in a buffer solution having a pH value of 10, at a pH value of 10, the carboxyl group dissociates and has a negative charge, and dissociates the quaternary ammonium group present inside the pores of the filler. It has a positive charge. Under such a condition, for example, the retention behavior when a sample containing a protein having an isoelectric point of 5 is injected will be described as follows. That is, if the ion exchange group inside the pores of the filler is a quaternary ammonium type, it is dissociated even in the buffer solution and has a positive charge as described above. When a low molecular anion approaches such a filler, ions are eliminated by the negative charge of the carboxyl group in the polymer (synthetic polymer substance) on the surface of the anion exchange resin. Since the positive charge of the exchange group is stronger, a low molecular anion enters inside the pore and is adsorbed by ion exchange. Further, when a protein having an isoelectric point smaller than that of the buffer approaches the filler, the sample protein also has a negative charge, and thus is subjected to ion exclusion due to the negative charge on the surface of the anion exchange resin.
Although it is possible that the protein is attracted by the positive charge of the anion exchange group inside the pore, the protein has a large molecular weight and cannot penetrate inside the pore, and cannot be adsorbed with the ion exchange group inside the pore. It is eluted. In practice, there is some hydrophobic adsorption and therefore some retention in the filler. In the case of low molecular weight cations, the negative charge of the carboxyl group in the polymer on the resin surface is ion-exchanged and adsorbed. I will. When a protein and a low molecular anion coexist in a sample, the above two phenomena occur simultaneously, and only the low molecular anion is retained by the packing material and separated.

 The above phenomena are summarized as follows. That is.

(A) When a protein and an enzyme coexist, ion exclusion occurs between the protein and the negative charge of the carboxyl group in the polymer on the resin surface. In addition, the pores of the filler cannot penetrate due to the large size of proteins and enzymes. For this reason, the negative charge on the surface is extremely small, and even a mobile phase having a low concentration is easily detached from the resin. Therefore, the holding force is small.

(B) In the case of a low molecular anion, the ion is excluded between the negative charge of the carboxyl group in the polymer on the resin surface.
In addition, it can penetrate into the pores of the filler, and ion exchanges with the anion exchange groups inside the pores. Therefore, the holding force is large.

(C) In the case of a low molecular cation, ion exchange adsorption is performed on the negative charge of the carboxyl group in the polymer on the resin surface. Further, it can penetrate into the pores of the filler, but is ion-excluded inside the pores. For this reason, since the negative charge on the surface is extremely small, even a mobile phase having a low concentration is easily detached from the resin. Therefore, the holding force is small.

<Effect of the Invention> According to the embodiment of the present invention described in detail above,
Anion exchange resin (filler) suitable for use in liquid chromatography (including ion chromatography) to quantitatively analyze the anions in samples packed with proteins and coexisting in chromatographic tubes. The manufacturing method is realized. In addition, the anion exchange resin produced in this way coats the surface of the base material with a synthetic polymer having a negative charge, so that low-molecular anions in samples containing proteins and the like can be removed without the influence of proteins. There is an advantage that it can be measured. Furthermore, since a sample containing a protein or the like can be directly injected and no extra operation such as washing is required, there is an advantage that the analysis conditions are simple and the measurement time is greatly reduced. In addition, the problem of reduced retention time caused by the apparent decrease in ion exchange capacity caused by the adsorption of proteins in the sample to the column packing material is eliminated, resulting in a chromatogram with good reproducibility. There is also the advantage that it can be done. Further, there is an advantage that the column performance is not degraded due to an increase in column pressure due to the adsorption of proteins and the like in the sample to the column packing material, thereby extending the life of the column.

[Brief description of the drawings]

FIG. 1 is a block diagram of the anion exchange resin of the present invention, FIG. 2 is a process explanatory view for explaining a method for producing the anion exchange resin of the present invention, and FIG. 3 is a diagram of a general ion chromatograph. FIG. A is an ion exchange resin, B is a polymer, C is an anion exchange group, P is a protein, S ⁺ is a cation, and S ⁻ is an anion.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) B01J 41/00 G01N 30/48 C08G 81/02

Claims (1)

(57) [Claims] 1. A spherical anion exchange resin for separating and quantifying anions in an aqueous solution in which a protein coexists, wherein the spherical body is bound to a surface and repels a charge that charges the protein. A polymer having a negative charge to be formed; micropores having a diameter smaller than the molecular diameter of the protein throughout the structure; and the pores bound to the inner surface of the micropores and entering the micropores. An anion exchange resin comprising: an anion exchange group that exchanges ions; and quantitatively analyzing the anion in a pH region where the protein has a negative charge.