JP2011169660A - Adsorbent for extractive separation and method for separating polar compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an adsorbent for a polar compound excellent in selectivity which can efficiently extract and separate the polar compound present in a sample to be inspected without being affected by impurities in a chemical analyzing method for performing the qualitative analysis and quantitative analysis of the polar compound in food or an environment sample. <P>SOLUTION: The polar compound is selectively and simply extracted and separated by developing hydrophilic interaction by chemically bonding an ampholytic ionic polymer 13, which has an imino or amino group and a carboxylic group in its molecule, to a hydrophilic base material 11 having a reactive functional group 12 which can be reacted with the imino or amino group to form a hydration layer on the surface of the adsorbent 10. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

In the chemical analysis and separation analysis for qualitative and quantitative analysis of organic compounds, the present invention extracts and separates polar compounds as measurement objects from samples such as the environment and food, and separates and removes non-measurement contaminants. The present invention relates to an adsorbent for extraction separation and a method for separating polar compounds.

Currently, several tens of thousands to 100,000 of chemical substances are produced on the scale of several hundred million tons per year. However, along with environmental pollution caused by these, there are concerns about adverse effects on humans and ecosystems. Further strengthening of the system is required. Substances used in large quantities in accordance with the Environmental Pollutant Release and Transfer Registration (PRTR) system based on the 1999 Act on Understanding the Emissions of Specific Chemical Substances to the Environment and Promoting Improvements in Management Efforts are being made to ascertain the volume and emissions to the environment. However, there are only a few hundred chemical substances subject to the PRTR system, and the actual situation regarding chemical substances that are used in small quantities, personal businesses, and households is not known. On the other hand, regarding foods, health effects due to the large use of residual agricultural chemicals and food additives are regarded as problems. In recent years, while “food safety and security” are screamed, the BSE outbreak in 2002 triggered a chain of beef disguise, a Chinese frozen vegetable problem, and an unregistered pesticide incident. The chain reaction of food damage occurred again, such as the Chinese frozen dumplings case, the contaminated rice case, and the melamine contamination case in China. Standards related to food ingredients (residue standards) have been established for some time, but with the revision of the Food Sanitation Law in 2003, certain amounts of pesticides, feed additives and veterinary drugs (such as agricultural chemicals) remained in foods. In May 2006, the positive list system was in effect to prohibit the sale of foods with pesticides remaining in excess of the amount.

Not only the environment and food, but also highly harmful compounds that should be monitored and regulated, accurate measurement is required even in trace amounts. Recent breakthroughs in instrumental analysis have enabled highly sensitive measurement of extremely small amounts of compounds at the ppb to ppt level. However, in the measurement of trace compounds present in the environment and foods, it is difficult to obtain reliable results due to the interference of a wide variety of contaminants. Moreover, there is a possibility that the contaminants behave as if they are the target compound, resulting in erroneous detection or a large positive error. In other words, the success or failure of the analysis largely depends on the pretreatment such as how to separate and remove highly contaminated substances. In order to cope with a wide variety of demands and the number of samples increasing year by year, advancement of pretreatment, labor saving, and speedup are important issues.

For pretreatment of an environment or food sample containing a large amount of contaminants, a method of preliminarily extracting by a solvent extraction method and then purifying by a solid phase extraction method is generally used. For example, in food analysis, with the enforcement of the positive list system, “Testing methods for substances that are pesticides, feed additives, or veterinary pharmaceutical ingredients remaining in food” has been issued by the Ministry of Health, Labor and Welfare as No. 0124001 Announced on January 24, 2005 (Non-Patent Document 1), then, “Regarding test methods for substances that are agricultural chemicals, feed additives or veterinary pharmaceutical ingredients remaining in food (partially revised)” It was notified on November 29, 2005 as Non-patent No. 1290002 (Non-patent Document 2). The test method related to the positive list system is “About the Use of Rapid Analysis Method for Residual Pesticides” notified on April 8, 1997 during the former Ministry of Health and Welfare (Heisei 43, Non-patent Document 3) Is based. In this notification test method, many simultaneous test methods using GC-MS and LC-MS, which are advanced analytical instruments, are employed. As shown in FIG. 7, the main steps in the sample pretreatment shown in Sanka No. 43 are performed in the order of solvent extraction → GPC fractionation → concentration. In some cases, direct extraction / concentration is performed from a pre-extraction solution using a solid-phase extraction method.

The basic principle of the solid-phase extraction method is based on the distribution of the solute between the two phases, similar to the solvent extraction method. However, the solid-phase extraction method is composed of a liquid phase and an insoluble solid phase. Extraction is performed using affinity for the solid phase. The solid-phase extraction method is widely used because it has been evaluated for environmental considerations such as excellent collection efficiency and reduction in the amount of organic solvent used. In addition, by changing the strength of the eluent stepwise, it is possible to sequentially elute the components extracted on the solid phase and perform fractionation / separation. The solid-phase extraction method is superior to the solvent extraction method in terms of selectivity and degree of purification, and has high adaptability for various purposes. As the solid phase extraction agent, a so-called reverse phase type solid phase extraction agent mainly utilizing hydrophobic interaction such as octadecyl group-bonded silica gel (generally referred to as ODS or C18) or polystyrene gel has become the mainstream. Since agrochemicals used from the past have many fat-soluble compounds, they can be easily extracted from a pre-extraction solution using these hydrophobic solid phase extractants and can be separated from contaminants. However, in recent years, agricultural chemicals with high biodegradability and low accumulation, that is, high hydrophilicity, are frequently used, and it is often difficult to cope with existing extraction techniques. In particular, the hydrophobic solid-phase extraction agent is not necessarily effective for extracting polar compounds because it extracts a measurement target compound by relying only on hydrophobic interaction.

In order to solve such a problem, an attempt has been made to promote the extraction of polar compounds by coexisting a functional group that exhibits a secondary interaction. Patent Document 1 discloses a solid-phase extraction agent using a copolymer of divinylbenzene and N-vinylpyrrolidone, and a wide range from polar compounds to hydrophobic compounds by using this solid-phase extraction agent. It is said that these compounds can be extracted simultaneously. Multicomponent simultaneous analysis has been achieved by fusing these solid-phase extraction agents with GC-MS or LC-MS. However, such solid-phase extraction agents have very low selectivity and specificity for highly water-soluble compounds such as highly polar compounds and ionic compounds. In fact, even in the above-mentioned notification test methods, for high-polarity pesticides etc., the simultaneous test method is not adopted and individual test methods are notified due to problems such as low extraction efficiency and different extraction conditions from other components. ing. In order to improve the extraction characteristics of the ionic compound, Patent Document 2 discloses a solid-phase extraction agent in which an ion exchange group is introduced into the copolymer of divinylbenzene and N-vinylpyrrolidone. By using this solid phase extractant, it became possible to increase the extraction rate of ionic compounds by the combined effect of ion exchange interaction and hydrophobic interaction. However, the extraction rate of compounds having low hydrophobicity and low ionicity and nonionic polar compounds is low, and it is difficult to obtain satisfactory extraction results.

In recent years, hydrophilic interaction has attracted attention as a novel separation mechanism of high performance liquid chromatography (Non-patent Document 4). The hydrophilic interaction is a complex interaction such as an interaction such as polarity or hydrogen bond based on the hydrophilic group of the adsorbent, or distribution to an aqueous phase held by the adsorbent. This separation system is composed of a polar stationary phase and a polar mobile phase such as acetonitrile, and separates polar compounds using affinity for the polar stationary phase. If this hydrophilic interaction can be used in the solid phase extraction method, it becomes easy to extract polar compounds that cannot be expected to have a high recovery rate with a hydrophobic solid phase extraction agent. Since a polar organic solvent is used as a solvent for the test solution, this is an effective technique for extracting a polar compound from a pre-extraction solution using a polar organic solvent frequently used in food analysis. In addition, since water or an aqueous salt solution is used for elution of polar compounds, it is an optimal pretreatment method when performing separation analysis by reverse phase high performance liquid chromatography such as ODS (C18). Adsorbents used for hydrophilic interaction include silica gel bonded with amino groups, amide groups, diol groups, etc., but the intrinsic affinity of these adsorbents for polar compounds is not necessarily high. In high-performance liquid chromatography, high affinity is not required because the separation is performed by continuous separation equilibrium, but in the solid-phase extraction method, the captured compounds are eluted by contaminant components or organic solvents contained in the sample. A high affinity is required so that it does not occur.

In response to this problem, Non-Patent Document 5 and Non-Patent Document 6 report on a solid-phase extraction agent in which sulfobetaine which is an amphoteric ion is introduced into a hydrophilic resin. According to this report, a hydration layer is formed on the surface of the adsorbent due to the high hydration power of the sulfobetaine group which is antichaotropic, and a polar compound is efficiently extracted into this hydration layer. In this document, it is said that the sulfobetaine type solid phase extractant showed the highest recovery rate with respect to various polar compounds in comparison with a commercially available solid phase extractant that expresses hydrophilic interaction. According to this document, it can be said that this solid phase extraction agent is an adsorbent / separation agent effective for extraction of polar compounds and high performance liquid chromatography of polar compounds. However, the zwitterion is supposed to behave as a nonionic compound by self-neutralization in the molecule, but when used for actual measurement, the counterion is bound and has a positive or negative charge. Conceivable. For example, when a large amount of sodium ions are present in the measurement sample, the sulfo group becomes a sodium type, so that it is positively charged based on the quaternary ammonium group. In particular, since the sulfobetaine group is highly ionic, depending on the compound to be measured, it is extracted and separated by a hydrophilic interaction with a strong ion exchange interaction or ion exclusion interaction added. In fact, according to the methods described in Non-Patent Document 5 and Non-Patent Document 6, the sulfo group has sodium as a counter ion. Furthermore, the reported introduction of functional groups is by no means high, and adjustment of the hydration layer (water retention amount and thickness of the hydration layer), that is, adjustment of hydrophobic interaction is not easy. Conceivable.

Patent Publication 2000-514704 Patent Publication 2002-517574

Ministry of Health, Labor and Welfare, Food Safety No. 012001, dated January 24, 2005 Ministry of Health, Labor and Welfare, Food Safety No. 1129002, Notification of November 29, 2005 Ministry of Health, Welfare No. 43, April 8, 1997 notice A. J. et al. Alpert: Journal of Chromatography, vol. 499, p. 177 (1990). T.A. Tsukamoto, A .; Yamamoto, W.H. Kamichitani and Y.K. Inoue: Chromatographia, vol. 70, p. 1525 (2009) T.A. Tsukamoto, M .; Yasuma, A .; Yamamoto, K .; Hirayama, T .; Kihou, S.A. Kodama and Y.K. Inoue: Journal of Separation Science, vol. 32, p. 3591 (2009)

The present invention has been made in view of the above-mentioned problems, and a zwitterionic polymer used in chemical analysis and separation analysis of polar compounds in a sample containing a large amount of impurities is a hydrophilic substrate. An object of the present invention is to provide an adsorbent suitable for extraction separation for a polar compound that expresses a hydrophilic interaction chemically bound to a surface.

As a result of intensive studies by the inventors of the present invention, a water-soluble zwitterionic polymer having a large number of imino groups or amino groups and carboxyl groups in the molecule is chemically bonded to a hydrophilic substrate, thereby It was found that an adsorbent capable of efficiently extracting and separating polar compounds by sexual interaction was obtained.

In the present invention, the water-soluble zwitterionic polymer chemically bonded to the hydrophilic substrate is a diallylamine-maleic acid copolymer represented by the following formula (1) or an allylamine represented by the following formula (2). -Maleic acid copolymers, and mixtures thereof.

The hydrophilic substrate used in the present invention is a hydrophilic copolymer obtained by a copolymerization reaction of a reactive monomer capable of reacting with an imino group or an amino group and a crosslinkable monomer, or an imino group. Or it is a hydrophilic copolymer which can introduce | transduce the functional group which can react with an amino group.

In the present invention, after binding a water-soluble zwitterionic polymer having a large number of imino groups or amino groups and carboxyl groups in the molecule represented by the formula (1) or (2) to a hydrophilic substrate, The introduced zwitterionic polymers are cross-linked using an epoxy compound.

In the present invention, a water-soluble zwitterionic polymer having a large number of imino groups or amino groups and carboxyl groups in the molecule represented by formula (1) or formula (2) is bonded to a hydrophilic substrate. In the crosslinking reaction with the epoxy compound, the zwitterionic polymer is further added together with the epoxy compound to perform crosslinking. By this method, the amount of water retention and the state of the hydrated layer are adjusted.

According to the present invention, a water-soluble zwitterionic polymer having a large number of imino groups or amino groups and carboxyl groups in the molecule is chemically bonded to a hydrophilic substrate, which is suitable for extraction and separation of polar compounds. It is possible to easily obtain an adsorbent. The adsorbent of the present invention is capable of selectively extracting and separating polar compounds from a pre-extraction solution of a sample such as an environment or food using a polar organic solvent by hydrophilic interaction and a highly hydrophilic polymer. Because hydrophobic compounds are not trapped by the high-level coating on the adsorbent surface, it is suitable as a solid phase extractant for pretreatment of polar compounds in samples such as the environment and food, and as a separating agent for high-performance liquid chromatography. Can be an adsorbent.

FIG. 1 is a conceptual diagram showing the binding state of the zwitterionic polymer of the adsorbent of the present invention and changes in the binding state of the zwitterionic polymer due to a crosslinking reaction. FIG. 2 is an example of a syringe-type solid phase extraction cartridge when the adsorbent of the present invention is used in the solid phase extraction method. FIG. 3 is an example of a Luer type solid phase extraction cartridge when the adsorbent of the present invention is used in the solid phase extraction method. FIG. 4 shows an operation flow when the adsorbent of the present invention is used in the solid phase extraction method. FIG. 5 is a graph comparing the extraction recovery rates of polar compounds in the zwitterionic polymer-bound adsorbents A, B, C of the present invention and the sulfobetaine-type adsorbents of the comparative examples. FIG. 6 is a chromatogram obtained by separating nucleobases by high performance liquid chromatography using the zwitterionic polymer-bound adsorbents D, E and F of the present invention. FIG. 7 is an explanatory diagram showing an operation flow of the rapid analysis method of pesticide residues in food.

In the present invention, an adsorbent suitable for the extraction and separation of polar compounds is obtained by chemically binding a water-soluble zwitterionic polymer having many imino groups or amino groups and carboxyl groups in the molecule to a hydrophilic substrate. To manufacture.

In the present invention, the water-soluble zwitterionic polymer bonded to the hydrophilic substrate is a diallylamine-maleic acid copolymer represented by the following formula (1) or an allylamine-maleic acid represented by the following formula (2). Copolymers are used, and these copolymers alone or a mixture of these copolymers may be bonded to the hydrophilic substrate. These zwitterionic polymers can be obtained, for example, by hydrolyzing a diallylamine or a copolymer obtained by copolymerization of allylamine and maleic anhydride. The abundance ratio of diallylamine or allylamine to maleic anhydride (n: m in formula (1) and formula (2)) is approximately 1: 1. Although it is difficult to accurately determine the molecular weight of these polymers, those having an average molecular weight of 5,000 to 100,000 are used in the present invention. These polymers are water-soluble, and a large number of water molecules are hydrated around each ionic group, and show high water retention. In general, a polymer does not extend linearly, but has a helical or spherical structure, and a large number of water molecules are retained inside these structures. In the hydrophilic interaction, the ionic group of the zwitterionic polymer can also contribute effectively to the extraction of the polar compound, but it is not preferable that the ion exchange interaction or the ion exclusion interaction is strongly expressed. For this reason, in the present invention, zwitterionic polymers having weak cationic and weak anionic functional groups are used.

Examples of the amphoteric polymer having a large number of imino groups or amino groups and carboxyl groups represented by the above formula (1) or formula (2) include aminoalkyl (meth) acrylates or copolymers of aminoalkylacrylamide and (meth) acrylic acid Can be given. Such amphoteric polymers can be obtained by copolymerization of aminoalkyl (meth) acrylate or aminoalkylacrylamide and (meth) acrylic acid, but aminoalkyl (meth) acrylate or aminoalkylacrylamide and (meth) acrylic. It can also be obtained by hydrolyzing the ester portion of the copolymer obtained by copolymerization with an acid alkyl ester. Examples of aminoalkyl (meth) acrylate or aminoalkylacrylamide include N, N-dimethylaminoethyl acrylate, N, N-dimethylaminoethyl methacrylate, N, N-diethylaminoethyl methacrylate, and N, N-dimethylaminopropyl acrylamide. It is done. Examples of the alkyl ester of (meth) acrylic acid include methyl methacrylate, methyl acrylate, ethyl methacrylate, and ethyl acrylate. Further, amphoteric polymers having a large number of imino groups or amino groups and carboxyl groups include glycidyl (meth) acrylate and (meth) acrylic acid or copolymers of (meth) acrylic acid alkyl esters with various amines. It is also possible to use a product obtained by reacting. When an alkyl ester of (meth) acrylic acid is used, the ester is hydrolyzed after the amine is reacted with the glycidyl group.

In the present invention, in order to express the hydrophilic interaction clearly, a hydrophilic material is also used for the substrate to which the zwitterionic polymer is bound. The hydrophilic substrate can be obtained by copolymerization of a hydrophilic monomer and a hydrophilic crosslinkable monomer by a known method. However, in order to chemically bond the zwitterionic polymer, a reaction is performed. It is necessary to have a functional group. Such a reactive substrate can be obtained by copolymerizing a reactive monomer and a crosslinkable monomer, or introducing a reactive functional group into a hydrophilic substrate by a post reaction. The binding of the zwitterionic polymer can be performed by a reaction with an imino group or an amino group, or a reaction with a carboxyl group. However, the ease of the binding reaction and the stability of the reaction product are limited. When considered, it is advantageous to utilize a reaction with an imino or amino group. Therefore, in the present invention, a substrate having a functional group capable of reacting with an imino group or an amino group is used as the reactive functional group.

One form of the hydrophilic substrate having a functional group capable of reacting with an imino group or an amino group includes a hydrophilic vinyl monomer having a functional group capable of reacting with an imino group or an amino group, and two or more vinyl groups. It is a crosslinkable polymer obtained by copolymerization with a hydrophilic crosslinkable monomer. Examples of the functional group of the hydrophilic vinyl monomer having a functional group that reacts with an imino group or an amino group include a halogenated alkyl group and an epoxy group. Examples of the reactive monomer having a halogenated alkyl group include 3-chloro-2-hydroxypropyl methacrylate, 3-chloro-2-hydroxypropyl acrylate, 2-chloroethyl methacrylate, 2-chloroethyl acrylate, and the like. Examples of the functional monomer having an epoxy group include glycidyl methacrylate and glycidyl acrylate. Examples of hydrophilic crosslinkable monomers having two or more vinyl groups copolymerizable with these monomers include ethylene dimethacrylate, diethylene glycol dimethacrylate, glycerin dimethacrylate, trimethylolpropane trimethacrylate, and neopentyl glycol trimethacrylate. Other examples include functional methacrylate monomers, crosslinkable monomers having a cyanuric acid skeleton such as triallyl isocyanurate and trimethallyl isocyanurate. In addition, a third vinyl monomer can be added in order to improve the physical properties such as hydrophilicity and hydrogen bonding properties of the hydrophilic substrate. Examples of vinyl monomers for improving physical properties include amide monomers such as N, N-dimethylacrylamide, N, N-diethylacrylamide, and morpholinoacrylamide, 2-hydroxyethyl methacrylate, glycerin methacrylate, neopentyl glycol methacrylate, and trimethylolpropane methacrylate. And methacrylate monomers such as N-vinylpyrrolidone.

In the hydrophilic substrate of the present invention, the amount of the vinyl monomer having a functional group that reacts with an imino group or an amino group is 20 to 80% by weight, preferably 30 to 80% by weight, based on the total vinyl monomer. In order to ensure sufficient hardness, at least 20% by weight or more of a hydrophilic crosslinkable monomer having two or more vinyl groups is used. Moreover, it is preferable to use 0-20 weight% of other vinyl monomers added in order to improve the physical properties, such as hydrophilic property and hydrogen bonding property of a hydrophilic base material.

In another embodiment of the hydrophilic substrate having a functional group capable of reacting with an imino group or an amino group, a functional group capable of reacting with an imino group or an amino group is introduced into a hydrophilic carrier having many hydroxyl groups by a chemical reaction. Can be obtained. Examples of the hydrophilic carrier having a large number of hydroxyl groups include hydrophilic hydrophilic carriers made of synthetic polymers such as polyvinyl alcohol-based crosslinkable resins and hydroxymethacrylate-based crosslinkable resins, and hydrophilic carriers made from polysaccharides such as cellulose and dextran. Etc. can be used. Examples of the functional group that reacts with the imino group or amino group include a halogenated alkyl group and an epoxy group. These functional groups can introduce an epoxy group by reacting a hydroxyl group of a hydrophilic carrier with epichlorohydrin or polyglycidyl ether. The introduced epoxy group can be converted into a halogenated alkyl group (chlorohydrin group) that reacts with an imino group or an amino group by treating with a hydrohalic acid as necessary. Further, the hydroxyl group of the hydrophilic carrier can be chlorinated by reaction with thionyl chloride in pyridine, and a halogenated alkyl group may be introduced by such a method.

In the present invention, since a high capturing ability is required, a porous substrate having a sufficient specific surface area is required. In addition, since the functional group to be bonded is a polymer, a pore size that allows the polymer to penetrate into the pores is necessary. Therefore, at the time of copolymerization of the vinyl monomer, the copolymerization is carried out in the presence of a solvent serving as a pore regulator that is compatible with the vinyl monomer and does not contribute to the polymerization reaction. The pore regulator is appropriately selected depending on the physical properties of the monomer used, but in general, aromatic hydrocarbons such as toluene and xylene, esters such as butyl acetate and dimethyl phthalate, amyl alcohol, octyl Slightly soluble alcohols such as alcohol and paraffins such as octane and dodecane are used and used in the range of 30 to 200 parts by weight, preferably 60 to 150 parts by weight, based on 100 parts by weight of the polymerizable monomer. Although the pore diameter and specific surface area of the porous substrate depend on the characteristics of the components to be captured and the coexisting components, in the present invention, the pore physical properties at the time of non-swelling are as follows: average pore diameter of 10 to 50 nm, specific surface area of 10 It is preferable to use one having a particle size of ˜800 m ² / g.

The particle size of the hydrophilic substrate used in the present invention can be arbitrarily set, but in general, the average particle size is 25 to 200 μm for solid phase extraction and 2 to 20 μm for high performance liquid chromatography. Things are used. Moreover, regarding the shape of the hydrophilic substrate, it may be an irregular crushing type or a spherical shape. When importance is attached to the flow characteristics and separation ability, the spherical shape is preferred. In this case, it is efficient to obtain a spherical copolymer by suspension polymerization.

FIG. 1 shows the binding state of the zwitterionic polymer of the adsorbent of the present invention and changes in the binding state of the zwitterionic polymer due to a crosslinking reaction. In the present invention, the zwitterionic polymer 13 is bonded to the hydrophilic substrate 11 by reacting with the imino group or amino group in the zwitterionic polymer 13 and the imino group or amino group in the hydrophilic substrate 11. It is carried out by reaction with a reactive functional group. In this case, the reaction with the carboxyl group in the zwitterionic polymer 13 may occur, so the reaction is performed under alkaline conditions. That is, a hydrophilic base material in which the zwitterionic polymer 13 to be introduced is dissolved in water or an aqueous solution of alcohol, dimethylformamide or the like, if necessary, and has a hydrophilic reactive functional group 12 in the solution. And is allowed to react at room temperature to 80 ° C. for 3 to 24 hours with stirring. Since there are many imino groups or amino groups in the zwitterionic polymer 13, it reacts with a plurality of reactive functional groups 12 in the hydrophilic substrate 11. Further, since there is entanglement between polymer chains, the adsorbent 10 of the present invention is in a state where the hydrophilic substrate 11 is covered with the zwitterionic polymer 13 as shown in FIG. 1a). . The introduction amount of the zwitterionic polymer 13 is adjusted by the amount and concentration of the zwitterionic polymer 13 and the reaction temperature. By the above reaction, it is possible to synthesize the adsorbent 10 having a hydrophilic interaction in which the zwitterionic polymer 13 having hydration property is bound.

As described above, since the zwitterionic polymer 13 bonded to the hydrophilic substrate 11 is layered and covers the hydrophilic substrate 11, a hydrated layer is formed on the surface of the hydrophilic substrate 11, In this state, hydrophilic interaction is expressed. Furthermore, in order to increase the amount of retained water and to express the interaction clearly, the degree of freedom of the zwitterionic polymer 13 bonded to the hydrophilic base material 11 is slightly limited, so that the adsorbent surface It is possible to further strengthen the hydration layer. As a method for limiting the degree of freedom of the polymer chains, a method of bonding the polymer chains by crosslinking is used. It is presumed that water clusters are formed in the three-dimensional space formed by this cross-linking and the hydration layer becomes stronger (FIG. 1b). In the figure, 14 is a crosslinking site. As a crosslinking method, after the zwitterionic polymer 13 is bonded to the hydrophilic substrate 11, an appropriate epoxy compound may be reacted under an alkaline condition. For example, in the case of an adsorbent in which a copolymer of diallylamine and maleic acid is bound, as shown in the following formula (3), the zwitterionic polymers 13 are cross-linked by the reaction between the remaining imino group and the epoxy compound. At the same time, a new hydroxyl group is generated by the ring-opening reaction of the epoxy group, and the hydrophilicity of the adsorbent is enhanced. Epoxy compounds include epihalohydrins such as epichlorohydrin, diglycidyl ethers of diols (glycols) such as ethylene glycol, propylene glycol, butanediol, and neopentyl glycol, glycerin, trimethylolpropane, pentaerythritol, sorbitol, etc. Polyglycidyl ethers of polyols can be used. Since the degree of crosslinking by these epoxy compounds affects the degree of freedom of the zwitterionic polymer 13 and the presence state of the surface, the thickness and state of the fixed hydrated layer change. When the degree of cross-linking is too high, the zwitterionic polymer 13 becomes a hard layer, so that it becomes difficult to form a hydrated layer. Since it is difficult to measure the degree of crosslinking with an epoxy compound, the amount of reagent at the time of reaction is adjusted. After introducing the zwitterionic polymer 13 into the hydrophilic substrate 11, the amount of introduced nitrogen is measured, and a crosslinking reaction is performed using an epoxy compound having 2 to 20 mol% of the amount of nitrogen. This crosslinking reaction produces a tertiary amino group in the case of a copolymer of diallylamine and maleic acid, and a secondary amino group in the case of a copolymer of allylamine and maleic acid. Since this tertiary or secondary amino group expresses an ion exchange interaction, the degree of crosslinking is adjusted depending on the characteristics of the test compound. By applying this technique, the test compound can be extracted and separated by the combined action of the hydrophilic interaction and the ion exchange interaction.

Furthermore, in order to thicken the hydration layer and to strongly develop the hydrophilic interaction, a method can be used in which the reaction is performed by adding the zwitterionic polymer 13 together with the epoxy compound during the crosslinking reaction. By using this method, as shown in FIG. 1c), the zwitterionic polymer 13 introduced by the primary reaction is crosslinked to form a layered structure having a clear three-dimensional structure, and at the same time, the added zwitterionic The polymer 13 can further undergo a crosslinking reaction to increase the thickness of the zwitterionic polymer layer. By such a method, it is possible to increase the amount of water retained and enhance the hydrophilic interaction. As a matter of course, since the amount and thickness of the zwitterionic polymer 13 to be bonded are increased by repeating this reaction, the degree can be adjusted by the number of repetitions.

The adsorbent 10 that exhibits the hydrophilic interaction produced according to the present invention is used for extraction of a polar compound by a solid phase extraction method or separation of a polar compound by high performance liquid chromatography.

When the adsorbent 10 is used in the solid-phase extraction method, for example, it is used by filling a solid-phase extraction cartridge (column) as shown in FIG. 2 or FIG. FIG. 2 shows a syringe-type solid phase extraction cartridge 20. The adsorbent 10 is filled in a cylindrical empty column body in which the lower frit 40 is inserted, and the upper frit 30 is inserted and fixed. FIG. 3 shows a lure type solid phase extraction cartridge 50. The adsorbent 10 is filled in an empty column body 51 in which the lower frit 40 is inserted. After the upper frit 30 is inserted, an empty column cap 52 is inserted and fixed. Here, 53 is an upper connection part that becomes an inlet of the solution, and 54 is a lower connection part that becomes an outlet of the solution.

An example of how to use such a solid phase extraction cartridge (column) will be described with reference to FIG. A measurement sample, for example, a food sample is homogenized and pre-extracted (adjusted) with a polar organic solvent miscible with water such as acetonitrile or acetone. After pre-extraction, filtration, dilution, etc. are performed as necessary.
For example, 10 mL each of acetonitrile, water, and 90% acetonitrile are passed through the cartridge for solid phase extraction in order. If necessary, acetone or tetrahydrofuran may be used instead of acetonitrile, and a buffer solution or an acid base may be used instead of water.
For example, 20 mL of the sample solution preliminarily extracted into the conditioned cartridge is passed, and the polar compound to be measured is captured by the adsorbent 10, concentrated, and extracted.
Thereafter, unnecessary components in the cartridge are washed with a polar organic solvent miscible with water containing water in a range in which the polar compound to be measured does not elute, for example, acetonitrile (acetone, tetrahydrofuran or the like, if necessary).
After washing, the polar compound is eluted with water (if necessary, a solution containing a buffer, acid base, or polar organic solvent). For the eluate containing a polar compound, the concentration in the solution is determined using high performance liquid chromatography, absorptiometry or the like.

When the adsorbent 10 of the present invention is used for high-performance liquid chromatography, an empty column having an appropriate inner diameter and length or internal volume is filled with a high-pressure slurry and used in a liquid chromatograph. As the mobile phase, a mixed solvent of a polar organic solvent miscible with water and water or a buffer solution, for example, a mixed solvent of acetonitrile and an aqueous ammonium acetate solution is used. If necessary, acetone, tetrahydrofuran, or the like can be used. In general, in hydrophilic interaction chromatography, the retention time increases as the concentration of the organic solvent in the mobile phase increases. Therefore, the separation is performed by adjusting the organic solvent concentration and setting conditions for obtaining appropriate separation. As a result, the polar compound to be measured is chromatographically separated by the adsorbent 10.

As described above, by using the adsorbent 10 combined with the zwitterionic polymer 13 of the present invention, it is possible to selectively and easily extract and separate polar compounds in food, feed, or environmental samples. . EXAMPLES Next, the present invention will be described with reference to examples, but the present invention is not limited to the examples.

Example 1 Synthesis of Zwitterionic Polymer Bond Adsorbent (Adsorbent for Extraction Separation) A (1) Synthesis of Hydrophilic Substrate L A hydrophilic substrate was prepared by an aqueous suspension polymerization method. A mixture prepared by dissolving 2 g of 2,2′-azobisisobutyronitrile in a mixed solution of 160 g of glycidyl methacrylate, 40 g of ethylene dimethacrylate, and 200 g of butyl acetate was added to 1000 mL of a 0.1% aqueous solution of methylcellulose and stirred using a stirrer. The blade was rotated at 400 rpm to disperse the oil layer. Thereafter, the dispersion (suspension) was heated, and a polymerization reaction was performed at 80 ° C. for 6 hours. The produced copolymer particles were collected by filtration and washed with water and methanol in this order. After air-drying for one day, classification was performed to obtain 75 g of a hydrophilic base material L of 45 to 90 μm. When the average particle size and dispersity (d75 / d25) of this hydrophilic substrate L were measured with a Beckman Coulter Multisizer 3 Coulter Counter, the average particle size was 64.5 μm and the dispersity was 1.26. Moreover, when the specific surface area and average pore diameter were measured with a Beckman Coulter SA3100 Surface Area Analyzer, the specific surface area was 31 m ² / g and the average pore diameter was 20 nm.

(2) Introduction of zwitterionic polymer 50 mL of diallylamine-maleic acid copolymer (Nittobo PSA-410C, concentration 40%) is added to a mixed solvent of 250 mL of water and 100 mL of 2-propanol to obtain a homogeneous solution. After that, 50 g of the hydrophilic substrate L obtained in (1) of Example 1 was added and stirred to obtain a uniform slurry. Further, 50 mL of 5M NaOH was added and reacted at 60 ° C. for 20 hours with stirring. After completion of the reaction, washing with 2-propanol, water, and methanol was performed in this order, followed by air drying overnight to obtain a zwitterionic polymer-bound adsorbent A. In order to determine the amount of the introduced zwitterionic polymer, the amount of nitrogen was 0.64 N% as measured by Perkin Elmar 2400 Series II CHNS / O Elemental Analyzer.

Example 2 Synthesis of Zwitterionic Polymer Bonded Adsorbent B 20 g of the zwitterionic polymer bonded adsorbent A obtained in Example 1 was dispersed in a mixed solution of 200 mL of water and 20 mL of 5M NaOH. A uniform slurry was obtained. Thereafter, 1 g of sorbitol polyglycidyl ether (manufactured by Nagase Chemtech, Denacol EX-611, average molecular weight of about 630) was added and reacted at 60 ° C. for 4 hours with stirring. After completion of the reaction, the mixture was washed with water and methanol in that order and air-dried overnight to obtain a zwitterionic polymer-bound adsorbent B. In order to determine the amount of the introduced zwitterionic polymer, it was measured by PerkinElmar 2400 Series II CHNS / O Elemental Analyzer, the nitrogen amount was 0.67 N%, and the amphoteric from the zwitterionic polymer-bound adsorbent A There was no desorption of the ionic polymer.

Example 3 Synthesis of Zwitterionic Polymer Bonded Adsorbent C 20 g of the zwitterionic polymer bonded adsorbent A obtained in Example 1 was dispersed in a mixed solution of 200 mL of water and 20 mL of 5M NaOH. A uniform slurry was obtained. Then, 50 mL of diallylamine-maleic acid copolymer (Nittobo PSA-410C, concentration 40%) was added to obtain a uniform slurry. Further, 1 g of sorbitol polyglycidyl ether (manufactured by Nagase Chemtech, Denacol EX-611, average molecular weight of about 630) was added and reacted at 60 ° C. for 4 hours while stirring. After completion of the reaction, water and methanol were washed in this order and air-dried overnight to obtain a zwitterionic polymer-bound adsorbent C. In order to determine the amount of the introduced zwitterionic polymer, the nitrogen amount was 0.92 N% as measured by PerkinElmar 2400 Series II CHNS / O Elemental Analyzer, which was amphoteric compared to the zwitterionic polymer-bound adsorbent A. The amount of binding of the ionic polymer was increased by about 50%.

(Comparative example) The sulfobetaine gel of the nonpatent literature 5 and the nonpatent literature 6 was synthesize | combined as a synthesis comparative example of a sulfobetaine type adsorbent. The synthesis method was performed with reference to Non-Patent Document 5 and Non-Patent Document 6. That is, a mixture of 1 g of 2,2′-azobisisobutyronitrile in a mixed solution of 50 g of glycidyl methacrylate, 10 g of N, N-dimethylacrylamide, 40 g of ethylene dimethacrylate, 40 g of butyl acetate and 60 g of 2-methylbutanol, It added to 1,000 mL of 0.2% polyvinyl alcohol (polymerization degree 500) aqueous solution, and the stirring blade was rotated at 250 rpm using the stirrer, and the oil layer was disperse | distributed. Thereafter, the dispersion (suspension) was heated, and a polymerization reaction was carried out at 70 ° C. for 7 hours. The produced copolymer particles were collected by filtration and washed with water and methanol in this order. After air-drying for one day, classification was performed to obtain 35 g of a hydrophilic substrate M having a thickness of 45 to 90 μm. When the average particle size of the hydrophilic substrate M was measured with a Beckman Coulter Multisizer 3 Coulter Counter, the average particle size was 64 μm. Moreover, when the specific surface area and average pore diameter were measured by Beckman Coulter SA3100 Surface Area Analyzer, the specific surface area was 84 m ² / g and the average pore diameter was 15.6 nm. Next, 15 mL of a 30% dimethylamine aqueous solution was added to 100 mL of a 10 wt% aqueous solution of sodium 2-bromoethanesulfonate and allowed to react at room temperature for 20 hours. Thereafter, the solution was passed through a chromatographic tube filled with a cation exchange resin (Dowex AG50-X4) and an anion exchange resin (Dowex AG1-X8) to remove excess impurities such as dimethylamine, and N, N-dimethylethane. A sulfonic acid aqueous solution was obtained. Thereafter, the crystals obtained by recrystallization were dried in a vacuum dryer at 40 ° C. To 1 g of the base material obtained by converting the hydrophilic base material M into a chlorohydrin type with 0.1 M hydrochloric acid, 1.5 g of N, N-dimethylethanesulfonic acid obtained by the above method and 20 mL of aqueous sodium hydroxide (pH 12.5) And reacted with stirring at 40 ° C. for 7 hours. After completion of the reaction, the mixture was washed with water and methanol in this order and dried to obtain a sulfobetaine-type adsorbent for comparison.

(Example 4) Extraction recovery comparison of polar compounds by solid phase extraction method 50 mg of the three zwitterionic polymer-bound adsorbents A, B and C obtained in Examples 1 to 3 A syringe type solid-phase extraction cartridge into which a filter with a pore size of 20 μm was inserted was filled, and a filter with a pore size of 20 μm was further inserted in the upper part. The sulfobetaine type adsorbent synthesized by the comparative example was also used as a cartridge in the same manner. The cartridge was conditioned by sequentially passing 10 mL of acetonitrile, 20 mL of pure water, and 10 mL of acetonitrile-water (9: 1) through the cartridge. Thereafter, 10 mL of a solution (concentration: 1 ppm) in which the test compound shown in Table 1 was dissolved in acetonitrile-water (9: 1) was passed through, and the test compound was extracted with each adsorbent. In Table 1, log Po / w is an octanol / water partition coefficient, and a smaller value means a compound that is easier to partition into water. Thereafter, after washing with 5 mL of acetonitrile, the test compound extracted to each adsorbent was eluted using 10 mL of pure water. Each eluate was measured using an ultraviolet-visible spectrophotometer, and the extraction recovery rate in each adsorbent was determined from the obtained absorbance and the absorbance of the test sample standard solution not subjected to the adsorption treatment. Table 1 shows the test compound, its solubility, and the water-octanol distribution ratio (log Po / w), and the structure is shown in Formula 4-9.

FIG. 5 shows the extraction recovery rates of the three types of zwitterionic polymer-bound adsorbents A, B, and C and the sulfobetaine type adsorbent of the comparative example. As is clear from FIG. 5, the three zwitterionic polymer-bound adsorbents A, B and C have higher extraction recovery rates in the order of C> B> A except for cytosine, and are zwitterionic during crosslinking. It can be seen that an adsorbent obtained by additionally reacting a polymer exhibits a higher hydrophilic interaction. The comparative sulfobetaine-type adsorbent showed high selectivity for adenine and uracil, and showed a slightly higher extraction recovery than the zwitterionic polymer-bound adsorbents B and C of the present invention. On the other hand, for the glycosides salicin and arbutin, the zwitterionic polymer-bonded adsorbents A, B and C of the present invention clearly showed a higher extraction recovery rate. From these results, it was found that the zwitterionic polymer-bound adsorbent of the present invention is suitable for extraction and separation of a wide range of water-soluble compounds. Regarding the nucleoside uridine to which the sugar is bound, the zwitterionic polymer-bonded adsorbents B and C of the present invention gave the same results as the sulfobetaine type adsorbent of the comparative example. Since the sulfobetaine type adsorbent of the comparative example shows high selectivity for uracil, it is considered that a high extraction recovery rate was obtained due to the selectivity of the base portion to uracil. In cytosine, it is considered that interactions other than hydrophilic interactions, probably an ion exclusion mechanism, are involved. This can be explained by the fact that the adsorbent recovery rate described in Non-Patent Document 5 and Non-Patent Document 6 is the lowest. This sulfobetaine-type adsorbent is presumed from the synthesis process that the counter ion of the sulfo group is a sodium ion, and as a result, the ion exclusion effect at the quaternary ammonium moiety was strongly expressed. On the other hand, since the zwitterionic polymer-bonded adsorbent of the present invention is a weak ionic functional group and has a low ion exclusion effect, it is considered that the extraction recovery rate was higher than that of the sulfobetaine type adsorbent. In particular, the non-crosslinked zwitterionic polymer-bound adsorbent A exhibited a higher extraction recovery rate, and therefore the ion exclusion effect was presumed to depend on the tertiary amino group produced by the crosslinking. . As is clear from these results, in the zwitterionic polymer-bound adsorbent of the present invention, hydrophilic interaction and ion exchange interaction can be achieved by changing the amount of crosslinking and binding according to the characteristics of the test compound. It can be seen that it can be easily changed.

Example 5 Synthesis of Zwitterionic Polymer Bonded Adsorbents D, E, and F (1) Synthesis of hydrophilic substrate N In a mixed solution of 80 g of glycidyl methacrylate, 120 g of ethylene dimethacrylate, and 200 g of butyl acetate, 2, 2 ′ -A mixture in which 2 g of azobisisobutyronitrile was dissolved was added to 1,000 mL of a 0.1% aqueous methylcellulose solution, and the oil layer was dispersed by a high speed rotation of a homomixer so that the center diameter of the oil droplets was about 10 μm. . Thereafter, the dispersion (suspension) was heated, and a polymerization reaction was performed at 80 ° C. for 6 hours. The produced copolymer particles were collected by filtration and washed with water and methanol in this order. After air drying for one day, classification was performed, and 80 g of hydrophilic base material N having an average particle diameter of about 10 μm was obtained using an air classifier. When the average particle size and dispersity (d75 / d25) of this hydrophilic substrate N were measured with a Beckman Coulter Multisizer 3 Coulter Counter, the average particle size was 10.1 μm and the dispersity was 1.16. Moreover, when the specific surface area and average pore diameter were measured by Beckman Coulter SA3100 Surface Area Analyzer, the specific surface area was 185 m ² / g and the average pore diameter was 12.9 nm.

(2) Introduction of zwitterionic polymer To the obtained hydrophilic base N, a diallylamine-maleic acid copolymer (Nittobo PSA-410C, concentration: 40%) under the same conditions as in (2) of Example 1 ), And then washed in the order of 2-propanol, water, and methanol, and then air-dried overnight to obtain a zwitterionic polymer-bound adsorbent D. The obtained zwitterionic polymer-bound adsorbent D was divided into three equal parts, one of which was sorbitol polyglycidyl ether (manufactured by Nagase Chemtech, Denacol EX-611, average molecular weight of about 1 under the same conditions as in Example 2. 630) was subjected to a cross-linking reaction, and a zwitterionic polymer-bound adsorbent E was obtained. Further, in the same manner as in Example 3, sorbitol polyglycidyl ether (manufactured by Nagase Chemtech, Denacol EX-611, A cross-linking reaction was carried out at an average molecular weight of about 630) to obtain a zwitterionic polymer-bound adsorbent F.

(3) Confirmation of separation of polar compound by high-performance liquid chromatography The zwitterionic polymer-bonded adsorbents D, E and F obtained in (2) of Example 5 were made of stainless steel having an inner diameter of 4.6 mm and a length of 150 mm. The chromatographic tube was filled and the nucleobases were separated by hydrophilic interaction chromatography. The measurement conditions are shown below.

High-performance liquid chromatography measurement conditions Mobile phase: acetonitrile-0.1M ammonium acetate (95 + 5)
Flow rate: 1.0 mL / min
Column temperature: 40 ° C
Detection: UV absorption detector (wavelength 250 nm)
Sample: thymine, uracil, adenine, cytosine, 10 ppm each
Sample injection volume: 40 μL

The measurement results are shown in FIG. In the figure, A is a zwitterionic polymer-bound adsorbent D, B is a zwitterionic polymer-bound adsorbent E, and C is a chromatogram of nucleobases using the zwitterionic polymer-bound adsorbent F. It is. As shown in FIG. 6, it was possible to satisfactorily separate nucleobases from the uncrosslinked and adsorbents for the additional crosslinking reaction. Under these conditions, thymine, uracil, adenine, and cytosine are eluted in this order. Generally, in the widely used reversed phase type separation agent, the elution order of these components is completely reversed. This result is not a separation mechanism based on hydrophobic interactions, but is separated by hydrophilic interactions. In comparison between the zwitterionic polymer-bound adsorbents, the elution time of thymine and uracil was almost the same, but the elution time of adenine was shorter in the order of D, E and F, and the elution time of cytosine was F, It was shorter in the order of E and D. The retention of adenine is considered to depend on the strength of hydrophilic interaction, that is, the strength of water retention of the zwitterionic polymer-bound adsorbent. On the other hand, regarding cytosine, as described in Example 4, it is presumed that the ion exclusion effect based on the tertiary amino group generated by the crosslinking reaction contributes. As is clear from these results, it was found that the zwitterionic polymer-bonded adsorbent of the present invention has a sufficient function even when it does not necessarily undergo a crosslinking reaction when used in high performance liquid chromatography. did. It can also be seen that the separation can be adjusted by easily changing the hydrophilic interaction and the ion exchange interaction by changing the amount of crosslinking and binding in accordance with the characteristics of the test compound.

According to the present invention, polar compounds in complicated contaminants can be selectively and easily extracted and separated by a simple method of chemically binding a water-soluble zwitterionic polymer to the surface of a hydrophilic substrate. Can be obtained. By using the adsorbent exhibiting the hydrophilic interaction of the present invention for solid phase extraction or high performance liquid chromatography, it becomes possible to qualitatively and quantify polar compounds with high accuracy. This simplifies the pretreatment of polar compounds, which conventionally required complicated pretreatment, or makes it possible to favorably separate polar compounds that were insufficiently separated in high performance liquid chromatography. Therefore, it becomes possible to measure a polar compound with higher reliability.

10: Adsorbent 11: Hydrophilic substrate 12: Reactive functional group (binding site)
13: Zwitterionic polymer 14: Cross-linked site 20: Syringe-type solid phase extraction cartridge (column)
30: Upper frit 40: Lower frit 50: Luer type solid phase extraction cartridge (column)
51: Empty column body 52: Empty column cap 53: Upper connection part 54: Lower connection part

Claims (7)

  An adsorbent for extraction and separation used for extraction and separation of polar compounds, a zwitterionic polymer having an imino group and a carboxyl group in the molecule, a zwitterionic polymer having an amino group and a carboxyl group in the molecule, An adsorbent for extraction / separation comprising a structure in which at least one of them is chemically bonded to a hydrophilic substrate. The zwitterionic polymer is a diallylamine-maleic acid copolymer represented by the following formula (1) or an allylamine-maleic acid copolymer represented by the following formula (2). 2. The adsorbent for extraction / separation according to 1.

The hydrophilic substrate is a hydrophilic copolymer obtained by a copolymerization reaction between a reactive monomer capable of reacting with an imino group or an amino group and a crosslinkable monomer, or reacted with an imino group or an amino group. The adsorbent for extraction and separation according to claim 1 or 2, wherein the adsorbent is a hydrophilic copolymer into which a functional group capable of being introduced can be introduced. The adsorption for extraction / separation according to any one of claims 1 to 3, wherein the zwitterionic polymer is bonded to the hydrophilic substrate and then the zwitterionic polymer is crosslinked with an epoxy compound. Agent. The adsorbent for extraction and separation according to claim 4, wherein when the zwitterionic polymer is crosslinked with the epoxy compound, the zwitterionic polymer is further added together with the epoxy compound to perform crosslinking. Measurement is performed by passing a test solution adjusted with a polar organic solvent miscible with water through an analytical column packed with the extraction / separation adsorbent according to any one of claims 1 to 5. After trapping and concentrating the target polar compound in the adsorbent, the adsorbent is washed with a polar organic solvent that is miscible with water containing water in a range where the polar compound to be measured does not elute, and then water, buffer A polar compound extraction method, wherein a polar compound to be measured is eluted from the adsorbent by using a liquid, acid-base polarity, or a solution obtained by adding an organic solvent thereto. A column in which the extraction / separation adsorbent according to any one of claims 1 to 5 is packed in a chromatographic tube for liquid chromatography is attached to a liquid chromatograph, and a polar organic solvent miscible with water and water or A method for separating a polar compound, wherein a mixed solvent with a buffer is passed as a mobile phase, and a polar compound to be measured is chromatographically separated with the adsorbent.

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