JP2007271310A - Method of analyzing hydrophobic organic compound and liquid chromatograph column - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analyzing method of a hydrophobic organic compound capable of clearly separating the hydrophobic organic compound by utilizing that an elution volume is different by the permutation of the solubilies of the hydrophobic organic compound to an aqueous solution and an eluent, hard to cause tailing, excellent in separation capacity, enabling the qualitative analysis or rough quantitative analysis of the hydrophobic organic compound, easy to obtain an analytical result of good reproducibility even if the preparatory detailed investigation of an analytical condition is not performed and markedly excellent in operability. <P>SOLUTION: The analyzing method of the hydrophobic organic compound includes a separation step for passing the aqueous solution containing the hydrophobic organic compound and the eluent through a column containing fine particles of a crosslinking polymer, which has a hydrophilic compound having a hydrophobic polymer chain bonded to its terminal and the hydrophilic unsaturated monomer crosslinked to the hydrophilic compound, as a filler and a detection step for detecting the hydrophobic organic compound passed through the column to be eluted to the eluent. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for analyzing a hydrophobic organic compound for analyzing a hydrophobic organic compound such as an endocrine disrupting substance in an aqueous solution, and a liquid chromatography column.

  Conventionally, organic compounds in the natural environment have attracted attention as a cause of environmental pollution, and recently as an exogenous endocrine disrupting substance (environmental hormone) that affects the ecosystem. For this reason, techniques for identifying species of organic compounds present in the environment are important. In addition, since there are several types of derivatives with similar chemical structures, such organic compounds can be efficiently separated and identified only by acquiring useful organic compounds for the development of new drugs such as pharmaceuticals and agricultural chemicals. This technology is essential for the development of various sciences.

As a method for analyzing a hydrophobic organic compound such as an environmental hormone dissolved in a minute amount in water, it is conventionally concentrated by liquid / liquid extraction described in (Non-patent Document 1) as a pretreatment, and this is subjected to high performance liquid chromatography. Analyzes were performed using analytical instruments such as gas chromatography.
In addition, as a method for analysis without performing pretreatment such as extraction and concentration, (Patent Document 1) states that “an alkylphenol in a water sample is converted into a fluorescent labeling agent under alkaline conditions and in the presence of a phase transfer catalyst”. Fluorescence labeling by sonication using, and removal of unreacted fluorescent labeling agent and by-products, followed by separation of fluorescently labeled alkylphenols by reverse phase liquid chromatography and chemifluorescence detection And a method for measuring alkylphenols having a process ".
Chemical Dictionary, Tokyo Chemical Co., Ltd., 1st edition, page 241 JP 2001-124750 A

However, the above conventional techniques have the following problems.
(1) The liquid / liquid extraction described in (Non-patent Document 1) is an established and useful technique. However, in the extraction of organic compounds present in low concentrations in water, a very large amount of organic solvent and multi-stage extraction are used. An extraction operation is required. In order to concentrate the target organic compound dissolved in a large amount of organic solvent, a larger amount of energy is required. For this reason, there existed the subject that the simplicity of operation, quickness, and energy saving lacked.
(2) In the technique disclosed in (Patent Document 1), alkylphenols to be detected must be fluorescently labeled by sonication using a fluorescent labeling agent under alkaline conditions and in the presence of a phase transfer catalyst. Therefore, there is a problem that the operation is complicated and lacks workability.
(3) Further, since a chemiluminescence detector is essential for detecting fluorescently labeled alkylphenols, it has a problem of lack of versatility.

The present invention solves the above-mentioned conventional problems, and the hydrophobic organic compound can be clearly separated by using the fact that the elution volume varies depending on the permutation of the solubility of the hydrophobic organic compound in the aqueous solution and the eluent. It is difficult to occur and has excellent resolution, and qualitative analysis and approximate quantitative analysis of hydrophobic organic compounds are possible. Reproducible analysis results can be easily obtained without requiring detailed analysis conditions in advance. An object of the present invention is to provide an excellent method for analyzing a hydrophobic organic compound.
In addition, the hydrophobic organic compound captured by passing through the eluent is easily eluted, and the hydrophobic organic compound can be separated by utilizing the difference in solubility. The purpose is to provide a column for liquid chromatography that has a wide range of selection of reagents and analysis conditions, and that it is easy to obtain reproducible analysis results without prior examination of detailed analysis conditions, and that is remarkably excellent in operability. And

In order to solve the above conventional problems, the method for analyzing a hydrophobic organic compound and the column for liquid chromatography of the present invention have the following constitutions.
In the method for analyzing a hydrophobic organic compound according to claim 1 of the present invention, a hydrophilic compound having a hydrophobic polymer chain bonded to a terminal, a hydrophilic unsaturated monomer cross-linked with the hydrophilic compound, A separation step of passing an aqueous solution containing a hydrophobic organic compound and an eluent through a column containing fine particles of a crosslinked polymer having a hydrophobicity, and the hydrophobicity eluted in the eluent that has passed through the column And a detection step of detecting an organic compound.
With this configuration, the following effects can be obtained.
(1) A crosslinked polymer in which a hydrophilic compound and a hydrophilic unsaturated monomer are cross-linked has a three-dimensional network structure, and a hydrophobic compound is present at the end of the hydrophilic compound in the network structure. Since the molecular chains are bonded, the hydrophilic compound forms a micelle structure by orienting the hydrophobic polymer chain inward in an aqueous solution, and a hydrophobic field (hereinafter referred to as hydrophobic field) with a size of several nanometers. A nano-domain). Hydrophobic organic compounds contained in an aqueous solution associate with hydrophobic nanodomains through hydrophobic interactions and are quickly captured. On the other hand, since the association between the hydrophobic nanodomain and the hydrophobic organic compound is relatively weak, the captured hydrophobic organic compound is easily eluted by passing an eluent having high affinity with the hydrophobic organic compound through the column. Therefore, using the fact that the elution volume varies depending on the permutation of the solubility of the hydrophobic organic compound in the aqueous solution and the eluent, the hydrophobic organic compound can be clearly separated and tailing is difficult to occur. Can be detected.
(2) Since sharp peaks of complicated hydrophobic organic compounds such as chemical modifications can be detected, the retention time of each of a plurality of peaks unique to the hydrophobic organic compound is obtained, and the known hydrophobic organic compound By collating with the retention time of the peak, a qualitative analysis of the hydrophobic organic compound can be performed.
(3) Since a sharp peak of a hydrophobic organic compound can be detected, the retention time of a peak of a hydrophobic organic compound having a known concentration is measured in advance, and the relationship between the concentration and the peak height or peak area is obtained. From the peak height or peak area when an unknown aqueous solution is analyzed under the same conditions by creating a calibration curve, an approximate quantitative analysis of the hydrophobic organic compound contained in the aqueous solution can also be performed by the calibration curve.
(4) In general, in liquid chromatography, separation is performed by utilizing the difference in the interaction between each component and the filler and eluent generated by adjusting the properties of the filler surface, the composition of the eluent, pH, etc. Since the analysis results differ depending on slight differences in conditions, it is necessary to examine the conditions of the filler and eluent in advance in advance, which requires complicated preliminary tests, but hydrophilic compounds and hydrophilic unsaturated monomers Columns packed with fine particles of cross-linked cross-linked polymers are relatively weak in association with hydrophobic organic compounds and are difficult to produce tailings, so there is a wide range of options for eluents and analytical conditions, etc. It is easy to obtain analytical results with good reproducibility without prior examination of analysis conditions, and the operability is remarkably excellent.

Here, as the hydrophobic polymer chain of the hydrophilic compound constituting the crosslinked polymer, at least one or more C _{6 to} C ₂₅ alkyl group or alkenyl group, preferably C _{8 to} C ₂₀ alkyl group or What has an alkenyl group is used, for example, a stearyl group, 2-ethyldecyl group, etc. are used.
As the number of carbon atoms of the alkyl group or alkenyl group is less than 8, the interaction with the hydrophobic organic compound is small and the bond is weakened, and the trapping ability of the hydrophobic organic compound tends to be lowered. There is a tendency that it is difficult to disperse uniformly with strong hydrophobicity. In particular, when the number is less than 6 or more than 25, these tendencies become remarkable, so that neither is preferable.

  As the hydrophilic compound, a known compound or a modified product thereof may be used as long as it is a linear polymer having an atomic group having a carbon-carbon double bond such as a vinyl group at the other end to which a hydrophobic polymer chain is bonded. Etc. can be used without particular limitation. For example, one obtained by esterifying polyethylene glycol monostearate having a hydroxyl group at one end with acrylic acid chloride is preferably used. A hydrophilic compound may be used independently and may use 2 or more types together.

As the hydrophilic unsaturated monomer constituting the crosslinked polymer, any monomer can be used without particular limitation as long as it is a monomer capable of obtaining a hydrophilic polymer by polymerization. For example, acrylic acid, methacrylic acid, maleic acid , Maleic anhydride, β-acryloyloxypropionic acid, fumaric acid, crotonic acid, itaconic acid, vinyl sulfonic acid, styrene sulfonic acid, 2- (meth) acrylamido-2-methylpropane sulfonic acid, 2- (meth) acryloylethane Hydrophilic unsaturated monomers containing acid groups such as sulfonic acid, 2- (meth) acryloylpropane sulfonic acid, sulfoethoxypolyethylene glycol mono (meth) acrylate and salts thereof; acrylamide, methacrylamide, N-ethyl (meth) Acrylamide, Nn-propyl (meth) acrylamide, N-iso Lopyl (meth) acrylamide, N, N-dimethyl (meth) acrylamide, 2-hydroxyethyl (meth) acrylate, 2-hydroxylpropyl (meth) acrylate, methoxypolyethylene glycol (meth) acrylate, polyethylene glycol mono (meth) acrylate, Nonionic hydrophilic unsaturated monomers such as vinylpyridine, N-vinylpyrrolidone, N-acryloylpiperidine, N-acryloylpyrrolidine; N, N-dimethylaminoethyl (meth) acrylate, N, N-diethylaminoethyl (meta 1) of cationic hydrophilic unsaturated monomers such as acrylate, N, N-dimethylaminopropyl (meth) acrylate, N, N-dimethylaminopropyl (meth) acrylamide and quaternary salts thereof, or the like 2 or more types Is used.
In addition, a hydrophilic unsaturated monomer that forms a hydrophilic polymer by hydrolysis of a functional group after polymerization, such as methyl (meth) acrylate, ethyl (meth) acrylate, and vinyl acetate, can also be used.
A predetermined amount of hydrophobic unsaturated monomer may be added to the hydrophilic unsaturated monomer. Examples of such hydrophobic unsaturated monomers include styrene, vinyl chloride, butadiene, isobutene, ethylene, propylene, stearyl (meth) acrylate, lauryl (meth) acrylate, and the like. These hydrophobic unsaturated monomers may be used in the range of 0 to 20 mol%, preferably 0 to 10 mol%, based on the total amount of the hydrophilic unsaturated monomer and the hydrophobic unsaturated monomer. it can.
As the content of the hydrophobic unsaturated monomer exceeds 10 mol% with respect to the total amount of the hydrophilic unsaturated monomer and the hydrophobic unsaturated monomer, the hydrophobicity becomes stronger and sufficient swelling is caused by water. There is a tendency that it is difficult to obtain, and if it exceeds 20 mol%, this tendency is remarkable, which is not preferable.

The crosslinked polymer preferably has a degree of crosslinking of 1 to 30%, preferably 3 to 15%, as measured by infrared absorption spectroscopy. This is because the crosslinked polymer is immersed in an aqueous solution to swell and become an aqueous gel, so that it has shape retention and can be used as a filler that passes through an eluent or an aqueous solution to capture a hydrophobic organic compound. .
As the degree of cross-linking becomes less than 3%, the shape retention is poor, and when the eluent passes, it tends to flow out of the column together with the eluent, and the pressure loss tends to increase. As the degree of cross-linking increases, the degree of swelling of the aqueous gel increases. Tends to be low and the trapping ability of the hydrophobic organic compound tends to decrease. In particular, if the content is lower than 1% or larger than 30%, these tendencies become remarkable, so that neither is preferable.

In the crosslinked polymer, the hydrophobic polymer chain preferably occupies 10 to 70% by weight, preferably 20 to 50% by weight of the total amount of the hydrophilic compound and the hydrophilic unsaturated monomer. It has a predetermined amount of hydrophobic nanodomains, and can achieve both compatibility with the hydrophobic organic compound in the crosslinked polymer and affinity with the aqueous solution, and capture a large amount of the hydrophobic organic compound in a short time. Because it can.
As the hydrophobic polymer chain becomes less than 20% by weight of the total amount of the hydrophilic compound and the hydrophilic unsaturated monomer, the affinity for the hydrophobic organic compound is poor and the trapping efficiency of the hydrophobic organic compound tends to decrease. As the amount exceeds 50% by weight, the hydrophobicity becomes stronger and it tends to be difficult to uniformly disperse the crosslinked polymer in the production of the crosslinked polymer. In particular, if the amount is less than 10% by weight or more than 70% by weight, these tendencies become remarkable, so that neither is preferable.

In the crosslinked polymer, the hydrophobic polymer chain preferably occupies 10 to 70% by weight, preferably 20 to 50% by weight of the hydrophilic compound. A hydrophilic compound can form micelles in an aqueous solution and form a predetermined amount of hydrophobic nanodomains that trap the hydrophobic organic compound, and the affinity of the crosslinked polymer with the hydrophobic organic compound and the aqueous solution This is because a large amount of hydrophobic organic compound can be captured in a short time.
As the proportion of the hydrophobic polymer chain occupies less than 20% by weight of the hydrophilic compound, the amount of the hydrophobic polymer chain decreases, the micelle formation ability decreases, and the ability to capture the hydrophobic organic compound tends to decrease. As the amount exceeds 50% by weight, the hydrophobicity becomes stronger and it tends to be difficult to uniformly disperse the crosslinked polymer in the production of the crosslinked polymer. In particular, if the amount is less than 10% by weight or more than 70% by weight, these tendencies become remarkable, so that neither is preferable.

The crosslinked polymer has a micelle formation process in which a hydrophilic compound having a hydrophobic polymer chain bonded to a terminal is associated with a hydrophilic compound having a critical micelle concentration or more in an aqueous solution to form a micelle, and the micelle is formed in the micelle formation process. And a polymerization reaction step in which a predetermined amount of a hydrophilic unsaturated monomer is added to the aqueous solution to form a crosslinked polymer.
By manufacturing as described above, micelles formed by associating a hydrophilic compound having a critical micelle concentration or higher in an aqueous solution in the micelle formation process are retained in the three-dimensional network structure of the crosslinked polymer in the polymerization reaction process. Therefore, the hydrophobic nanodomains formed by the micelles can be dispersed and held in the crosslinked polymer, and a crosslinked polymer excellent in the trapping efficiency of the hydrophobic organic compound can be produced.

  Here, as the aqueous solution used in the micelle formation step, an aqueous solution such as ion-exchanged water is used. If micelles can be formed, a mixed medium such as water and alcohol can also be used.

  In the polymerization reaction step, the crosslinked polymer is a known method such as a radical polymerization method, a redox polymerization method, a living radical polymerization method, a method of irradiating radiation or electron beam, a method of irradiating light in the presence of a photosensitizer, Can be used. Of these, the radical polymerization method is preferably used. This is because the versatility is extremely high for copolymerization with other monomers.

  As a crosslinking agent used in the polymerization reaction step, a water-soluble compound having at least two functions and capable of copolymerizing a water-soluble unsaturated monomer and a hydrophilic compound, for example, N, N′-methylenebisacrylamide, N, N ′. -Bisacrylamides such as methylenebismethacrylamide; long-chain diacrylates such as ethylene glycol di (meth) acrylate and polyethylene glycol di (meth) acrylate; divinylbenzene and the like are preferably used. These can be used alone or in combination of two or more.

The initiator in the radical polymerization method is not particularly limited as long as it is water-soluble and can be used. For example, ammonium persulfate, potassium persulfate, hydrogen peroxide, tert-butyl hydroperoxide and the like; redox initiators such as sulfite, hydrogen sulfite and ceric ammonium nitrate; 2,2′-azobis-2-amidino Propane hydrochloride, 2,2'-azobis-2,4-dimethylvaleronitrile, azo compounds such as 4,4'-azobis-4-cyanovaleric acid and its salts, and the like can be used. These initiators can be used alone or in combination of two or more.
The initiator is used in an amount of 0.01 to 10% by weight, preferably 0.01 to 8% by weight, based on the total amount of the hydrophilic compound and the hydrophilic unsaturated monomer. When the amount of the initiator is less than 0.01% by weight, the polymerization efficiency is extremely lowered and the productivity is insufficient. When the amount exceeds 8% by weight, the molecular weight of the cross-linked polymer produced tends to be small, and the strength as a hydrophobic organic compound trapping material tends to be maintained. Particularly, when the amount exceeds 10% by weight, this tendency is remarkable. Therefore, it is not preferable.

The filler is obtained by pulverizing the crosslinked polymer with a pulverizer such as a ball mill or a universal mill to produce fine particles. Further, in the polymerization reaction step, the aqueous solution can also be obtained by stirring or emulsion polymerization using a homomixer, a propeller stirrer or the like to produce fine particles.
A filler having a diameter of 1 μm to 1 mm is preferably used. If the particle size of the packing material is smaller than 1 μm, it will be easy to flow out from the packed column, and if a filter with a small opening diameter is provided in the column in order to prevent outflow, a large loss will occur when the eluent passes. Absent. When the particle diameter of the filler is larger than 1 mm, the surface area is reduced and the trapping ability of the hydrophobic organic compound is lowered, which is not preferable. The shape of the filler is not limited to a spherical shape, and various shapes can be used.

  As the material and size of the column, the same materials as those used in high performance liquid chromatography (HPLC) can be used. For example, those having an inner diameter of 1 to 20 mm are preferable, and those having an inner diameter of 1 to 5 mm are preferably used. The length is preferably 5 to 40 cm, particularly preferably 10 to 20 cm. A material made of stainless steel or synthetic resin can be used without any particular limitation.

As the aqueous solution, water, a mixed solution of water and alcohol, or the like is used. The aqueous solution may contain an inorganic salt or the like. As such an aqueous solution, tap water, purified water, river water, lake water, ground water, rainwater, drainage water, brackish water, seawater and the like are used.
When a mixed solution of water and alcohol is used as the aqueous solution, an aqueous solution having an alcohol concentration of 40% by volume or less is used. This is because as the alcohol concentration becomes higher than 40% by volume, the association of the hydrophilic compounds forming the hydrophobic nanodomains is inhibited and the hydrophobic nanodomains cannot be maintained in the crosslinked polymer.

  Examples of hydrophobic organic compounds that can be analyzed include phenols and phenol derivatives such as phenol, nitrophenol, alkylated phenol, and bisphenol A, aromatic hydrocarbon compounds such as benzene, toluene, and xylene, organic acids such as carboxylic acids, ketones, Examples thereof include nitrile compounds such as aldehydes, esters, organic amines, aromatic amines, acetonitrile, and benzonitrile, and endocrine disrupting substances such as chloroform, dichloroethane, estradiol-17-glucyl chloride, and estradiol-3-glucyl chloride.

  As the eluent, one having a solubility of the hydrophobic organic compound in the eluent larger than that in the aqueous solution is used. This is because the separation can be performed using the permutation of the solubility of the hydrophobic organic compound in the aqueous solution and the eluent. Such an eluent can be selected from solvents usually used in column chromatography and mixtures thereof. For example, one or more water-soluble organic solvents such as methanol, ethanol, acetone, and tetrahydrofuran are used. A mixture of these organic solvents and water is used. In particular, ethanol is preferably used. This is because the miscibility with water is good and the solubility of the hydrophobic organic compound is relatively high.

The flow rate of the eluent in the column is not particularly limited as long as the hydrophobic organic compound can be separated. For example, 0.5 to 10 mL / min, preferably 1 to 5 mL / min is suitable.
The temperature of the column is not particularly limited as long as the hydrophobic organic compound can be separated, but for example, 0 to 50 ° C, preferably 15 to 30 ° C is suitable.

  The detector for detecting the hydrophobic organic compound in the detection step is not particularly limited as long as it is usually used in column chromatography. For example, a differential refractive index detector, an ultraviolet absorption detector, a fluorescence detector, a photo detector A diode array detector or the like is used.

The invention according to claim 2 of the present invention is the method for analyzing a hydrophobic organic compound according to claim 1, wherein the solubility of the hydrophobic organic compound in the eluent is larger than the solubility in the aqueous solution. It has the structure of.
With this configuration, in addition to the operation obtained in the first aspect, the following operation can be obtained.
(1) Since the solubility of the hydrophobic organic compound in the eluent is larger than the solubility in the aqueous solution, the hydrophobicity is determined by utilizing the elution volume depending on the permutation of the solubility of the hydrophobic organic compound in the aqueous solution and the eluent. Organic compounds can be separated and analyzed.

Invention of Claim 3 of this invention is the analysis method of the hydrophobic organic compound of Claim 1 or 2, Comprising: The said hydrophobic organic compound is a phenol and a phenol derivative, an aromatic hydrocarbon compound, organic It has the structure which is 1 type or multiple types of an acid, a nitrile compound, and an endocrine disrupting substance.
With this configuration, in addition to the operation obtained in the first or second aspect, the following operation can be obtained.
(1) Since hydrophobic organic compounds are phenols and phenol derivatives, aromatic hydrocarbon compounds, organic acids, nitrile compounds, and endocrine disruptors, they can be used to identify pollutants in the environment and to develop new drugs such as pharmaceuticals and agricultural chemicals. Useful chemical substances can be easily and quickly identified.

The column for liquid chromatography according to claim 4 of the present invention comprises a hydrophilic compound having a hydrophobic polymer chain bonded to a terminal thereof, and a hydrophilic unsaturated monomer cross-linked with the hydrophilic compound. It has a structure filled with fine particles of a crosslinked polymer.
With this configuration, the following effects can be obtained.
(1) A crosslinked polymer in which a hydrophilic compound and a hydrophilic unsaturated monomer are cross-linked has a three-dimensional network structure, and a hydrophobic compound is present at the end of the hydrophilic compound in the network structure. Since molecular chains are bonded, hydrophilic compounds form a micellar structure by orienting hydrophobic polymer chains inward in aqueous solution, forming hydrophobic nanodomains with a size of several nanometers To do. Hydrophobic organic compounds contained in an aqueous solution associate with hydrophobic nanodomains through hydrophobic interactions and are quickly captured. On the other hand, since the association between the hydrophobic nanodomain and the hydrophobic organic compound is relatively weak, the captured hydrophobic organic compound is easily eluted by passing through an eluent having high affinity with the hydrophobic organic compound. The hydrophobic organic compound can be clearly separated by utilizing the fact that the elution volume varies depending on the permutation of the solubility of the hydrophobic organic compound in the aqueous solution and the eluent.
(2) Since fine particles of a crosslinked polymer in which a hydrophilic compound and a hydrophilic unsaturated monomer are cross-linked are packed, the association with the hydrophobic organic compound is relatively weak and tailing is unlikely to occur. The range of selection of agents and analysis conditions is wide, and it is easy to obtain analysis results with good reproducibility without prior examination of detailed analysis conditions.

  Here, since the hydrophobic polymer chain, the hydrophilic compound, the hydrophilic unsaturated monomer, the crosslinked polymer, and the fine particles are the same as those described in claim 1, the description thereof is omitted.

As described above, according to the method for analyzing a hydrophobic organic compound and the column for liquid chromatography of the present invention, the following advantageous effects can be obtained.
According to the invention of claim 1,
(1) The crosslinked polymer forms a micelle structure and forms a hydrophobic field (hydrophobic nanodomain) having a size of several nanometers. The hydrophobic organic compound contained in the aqueous solution is composed of a hydrophobic nanodomain and a hydrophobic nanodomain. Hydrophobic organic compounds are easily eluted by passing eluents with high affinity to hydrophobic organic compounds through the column while weakly associating weakly by sexual interactions. By utilizing the fact that the elution volume varies depending on the permutation of the solubility of the compound in the aqueous solution and the eluent, it is possible to provide a method for analyzing the hydrophobic organic compound that can clearly separate the hydrophobic organic compound and does not produce tailing and has excellent resolution. .
(2) Since a sharp peak of a hydrophobic organic compound can be detected, the retention time of each of a plurality of peaks unique to the hydrophobic organic compound is obtained and compared with the retention time of a known hydrophobic organic compound peak. By doing so, a method for analyzing a hydrophobic organic compound capable of qualitative analysis of the hydrophobic organic compound can be provided.
(3) Since a sharp peak of a hydrophobic organic compound can be detected, the retention time of a peak of a hydrophobic organic compound having a known concentration is measured in advance, and the relationship between the concentration and the peak height or peak area is obtained. Hydrophobic organic that can also perform approximate quantitative analysis of hydrophobic organic compounds contained in an aqueous solution using a calibration curve from the peak height or peak area when an unknown aqueous solution is analyzed under the same conditions by creating a calibration curve A method for analyzing a compound can be provided.
(4) Since a column filled with fine particles of a crosslinked polymer in which a hydrophilic compound and a hydrophilic unsaturated monomer are cross-linked is used, the association with the hydrophobic organic compound is relatively weak and tailing occurs. Since it is difficult to select eluents and analysis conditions, it is easy to obtain reproducible analysis results without performing detailed examination of detailed analysis conditions. Can provide a method.

According to invention of Claim 2, in addition to the effect of Claim 1,
(1) Since the solubility of the hydrophobic organic compound in the eluent is larger than the solubility in the aqueous solution, the hydrophobicity is determined by utilizing the elution volume depending on the permutation of the solubility of the hydrophobic organic compound in the aqueous solution and the eluent. A method for analyzing a hydrophobic organic compound capable of separating and analyzing the organic compound can be provided.

According to invention of Claim 3, in addition to the effect of Claim 1 or 2,
(1) Since hydrophobic organic compounds are phenols and phenol derivatives, aromatic hydrocarbon compounds, organic acids, nitrile compounds, and endocrine disruptors, they can be used to identify pollutants in the environment and to develop new drugs such as pharmaceuticals and agricultural chemicals. It is possible to provide a method for analyzing a hydrophobic organic compound, which can easily and quickly identify useful chemical substances.

According to invention of Claim 4,
(1) Hydrophobic organic compounds associate with hydrophobic nanodomains by hydrophobic interaction and are quickly captured, and the association between hydrophobic nanodomains and hydrophobic organic compounds is relatively weak, and thus is captured by the eluent. Thus, the hydrophobic organic compound can be easily eluted, and the hydrophobic organic compound can be separated using the difference in solubility, so that tailing hardly occurs and a liquid chromatograph column excellent in separation performance can be provided.
(2) Since fine particles of a crosslinked polymer in which a hydrophilic compound and a hydrophilic unsaturated monomer are cross-linked are packed, the association with the hydrophobic organic compound is relatively weak and tailing is unlikely to occur. Therefore, it is possible to provide a liquid chromatograph column that has a wide range of selection of reagents, analysis conditions, and the like, and can easily obtain a reproducible analysis result without prior examination of detailed analysis conditions.

Hereinafter, the best mode for carrying out the present invention will be described with reference to the drawings.
(Embodiment 1)
FIG. 1 is a configuration diagram of an apparatus for analyzing a hydrophobic organic compound using a liquid chromatograph column according to Embodiment 1 of the present invention.
In the figure, 1 is an analyzer for a hydrophobic organic compound, 2 is a container, 3 is an eluent which is a water-soluble organic solvent such as ethanol or tetrahydrofuran, or a mixed solvent of an organic solvent and water, contained in the container 2. Is a liquid flow path whose one end is immersed in the eluent 3, 5 is a pump which is disposed in the liquid flow path 4 and sends the eluent 3 from the liquid flow path 4 to a liquid chromatograph column 7 which will be described later. A sample injection section 7 disposed in the liquid flow path 4 downstream of the liquid channel 4 is a liquid chromatograph column having one end connected to the other end of the liquid flow path 6. The liquid chromatograph column 7 is filled with fine particles of a crosslinked polymer having a hydrophilic compound having a hydrophobic polymer chain such as a stearyl group bonded to the terminal and a hydrophilic unsaturated monomer such as acrylamide crosslinked. As filled. 8 is a thermostatic chamber containing the liquid chromatograph column 7, 9 is a liquid flow path having one end connected to the other end of the liquid chromatograph column 7, and 10 is a differential refractive index disposed in the liquid flow path 9. Detectors such as detectors, ultraviolet absorption detectors, fluorescence detectors, and photodiode array detectors.

Hereinafter, a method for analyzing a hydrophobic organic compound will be described using the analyzer 1 configured as described above.
After keeping the inside of the column thermostat 8 at a predetermined temperature and holding the liquid chromatograph column 7 at a predetermined temperature, an aqueous solution containing a hydrophobic organic compound as a sample is injected from the sample injection unit 6. The pump 5 is driven to send the eluent 3 to the liquid flow path 4, and the sample (aqueous solution) injected from the sample injection section 6 by the eluent 3 is passed to the liquid chromatograph column 7. Since the liquid chromatographic column 7 is filled with a filler of a crosslinked polymer that forms a micelle structure and forms a hydrophobic field (hydrophobic nanodomain), the hydrophobic organic compound contained in the aqueous solution is: The hydrophobic nanodomain is associated with the hydrophobic interaction and rapidly captured, but since the association between the hydrophobic nanodomain and the hydrophobic organic compound is relatively weak, the eluent 3 having a high affinity with the hydrophobic organic compound is formed. Elution is easy. Since the elution volume varies depending on the permutation of the solubility of the hydrophobic organic compound in the aqueous solution and the eluent 3, the hydrophobic organic compound is separated in the liquid chromatographic column 7 (the separation step). Next, in the detection step, the hydrophobic organic compound can be analyzed by detecting the separated hydrophobic organic compound with the detector 10.

According to the method for analyzing a hydrophobic organic compound as described above, the following action can be obtained.
(1) An aqueous solution containing a hydrophobic organic compound is applied to a liquid chromatograph column 7 filled with a filler of a crosslinked polymer that forms a micelle structure and forms a hydrophobic field (hydrophobic nanodomain). 3, the hydrophobic organic compound contained in the aqueous solution associates with the hydrophobic nanodomains by hydrophobic interaction and is quickly captured, while the association between the hydrophobic nanodomains and the hydrophobic organic compound is Since the hydrophobic organic compound captured by the eluent 3 is relatively weak because it is relatively weak, the elution volume varies depending on the permutation of the solubility of the hydrophobic organic compound in the aqueous solution and the eluent. The organic organic compound can be clearly separated and tailing hardly occurs, and the sharp peak of the hydrophobic organic compound can be detected by the detector 10.
(2) Since a sharp peak of a hydrophobic organic compound can be detected, the retention time of each of a plurality of peaks unique to the hydrophobic organic compound is obtained and compared with the retention time of a known hydrophobic organic compound peak. By doing so, a qualitative analysis of the hydrophobic organic compound can be performed.
(3) Since a sharp peak of a hydrophobic organic compound can be detected, the retention time of a peak of a hydrophobic organic compound having a known concentration is measured in advance, and the relationship between the concentration and the peak height or peak area is obtained. From the peak height or peak area when an unknown aqueous solution is analyzed under the same conditions by creating a calibration curve, an approximate quantitative analysis of the hydrophobic organic compound contained in the aqueous solution can also be performed by the calibration curve.
(4) The liquid chromatographic column 7 packed with a bridging polymer packing material that forms a micelle structure and forms a hydrophobic field (hydrophobic nanodomain) has a relatively weak association with a hydrophobic organic compound and tails. Therefore, it is easy to obtain an analysis result with good reproducibility without reexamination of detailed analysis conditions, and the operability is remarkably excellent.

Further, according to the liquid chromatograph column 7 in the first embodiment of the present invention as described above, the following operation is obtained.
(1) Since it is filled with a filler of a crosslinked polymer that forms a micelle structure and forms a hydrophobic field (hydrophobic nanodomain), an aqueous solution containing a hydrophobic organic compound is passed by a dissociator 3 The hydrophobic organic compound is weakly associated with and captured by the hydrophobic nanodomain, and is easily eluted by the eluent 3 having a high affinity with the hydrophobic organic compound. Utilizing the fact that the elution volume varies depending on the permutation of the solubility in the aqueous solution and the eluent, the hydrophobic organic compound can be clearly separated, and tailing hardly occurs and the separation performance is excellent.
(2) Since fine particles of a crosslinked polymer in which a hydrophilic compound and a hydrophilic unsaturated monomer are cross-linked are packed, the association with the hydrophobic organic compound is relatively weak and tailing is unlikely to occur. The range of selection of agents and analysis conditions is wide, and it is easy to obtain analysis results with good reproducibility without prior examination of detailed analysis conditions.

Hereinafter, the present invention will be specifically described by way of examples. The present invention is not limited to these examples.
Example 1
Polyethylene glycol monostearate (manufactured by Tokyo Chemical Industry Co., Ltd.) having a number average molecular weight of 2,000 having a non-hydrophobized terminal hydroxylated in N, N-dimethylformamide as a solvent placed in a glass container with a stirrer, acrylic acid Chloride (manufactured by Tokyo Chemical Industry Co., Ltd.) and 4-N, N-dimethylaminopyridine were added at a molar ratio of 1: 1.02: 1, and the mixture was stirred at room temperature in a stream of dry nitrogen for 24 hours. A hydrophilic compound (polyethylene glycol monostearate; hereinafter referred to as M1) was obtained. By analyzing using high resolution nuclear magnetic resonance (JNM-ECP500 manufactured by JEOL), it was confirmed that the end of the obtained hydrophilic compound (M1) was acrylated.
1 mol which becomes more than the critical micelle concentration of the manufactured hydrophilic compound (M1) was dissolved in water to form micelles (micelle formation step).
Next, 1 mol of acrylamide (hereinafter referred to as AAM) as a hydrophilic unsaturated monomer, 0.1 mol of methylene bisacrylamide (hereinafter referred to as MBA) as a crosslinking agent, and potassium persulfate (as an initiator) Hereinafter, 0.01 mol of KPS) and 0.01 mol of tetramethylethylenediamine (hereinafter referred to as TMEDA) as a catalyst were added and polymerized at a reaction temperature of 40 ° C. to form a three-dimensional network crosslinked polymer. (Polymerization reaction step). The crosslinked polymer retains the micelle structure formed in the aqueous solution with the hydrophilic compound (M1) as it is, and the micelle structure is retained in the gel obtained by swelling the crosslinked polymer with water. This was confirmed by a small angle X-ray scattering method. The degree of crosslinking measured by infrared absorption spectroscopy was 10%.
The crosslinked polymer produced as described above was pulverized by a universal mill and then classified to a particle size of 20 to 100 μm using a sieve to obtain the filler of Example 1.
The obtained filler was slurried with ion-exchanged water and packed into a stainless steel column having a length of 10 cm and an inner diameter of 5 mm, whereby the liquid chromatograph column of Example 1 was obtained.

(Comparative Example 1)
A column for liquid chromatography of Comparative Example 1 is packed by using a silica gel (Wakogel, C-300E, manufactured by Wako Pure Chemical Industries), which is generally used in liquid chromatography, as a packing material in a column having the same shape as Example 1. Got.

(Qualitative analysis of sample)
The liquid chromatograph columns of Example 1 and Comparative Example 1 were mounted on the analyzer 1 described in Embodiment 1, and samples were analyzed under the following conditions. As a detector, an ultraviolet absorption detector (wavelength 280 nm) was used.
Eluent: ethanol, column thermostat temperature: 25 ° C., eluent flow rate: 1 mL / min, sample injection volume: 100 μL.
Sample: 1 ppm of bisphenol A (hereinafter referred to as BPA), 1 ppm of biphenol (hereinafter referred to as BP), 1 ppm of dodecylphenol (hereinafter referred to as DP), 1,2,4-trichlorobenzene (hereinafter referred to as TCB) An aqueous solution in which 1 ppm is dissolved in ion-exchanged water.
The analysis results are shown in FIG. FIG. 2 is an elution curve obtained by elution analysis of the above sample using the liquid chromatograph column of Example 1 and Comparative Example 1. The horizontal axis indicates elution time (retention time) (minutes), and the vertical axis indicates ultraviolet rays. The absorption peak height is shown.
FIG. 2 shows that tailing occurs in Comparative Example 1, the detected peak is broad, and the four types of hydrophobic organic compounds are not clearly separated. On the other hand, in Example 1, four clear sharp peaks could be confirmed.
By obtaining the retention times of known dodecylphenol (DP), bisphenol A (BPA), biphenol (BP), and 1,2,4-trichlorobenzene (TCB) peaks in advance and collating them with this known data Qualitative analysis can be performed. As a result of collation, it was found that the four peaks were dodecylphenol (DP), bisphenol A (BPA), biphenol (BP), and 1,2,4-trichlorobenzene (TCB) in order from the left.

(Quantitative analysis of hydrophobic organic compounds)
2 ppm bisphenol A (BPA) aqueous solution, 5 ppm bisphenol A (BPA) aqueous solution, 8 ppm bisphenol A (BPA) aqueous solution, 10 ppm bisphenol under the same conditions as above using the analyzer equipped with the liquid chromatograph column of Example 1. Elution analysis was performed using A (BPA) aqueous solution as a sample, and the peak height detected by the detector was determined. By plotting the peak height (or peak area) against the concentration of the hydrophobic organic compound, a calibration curve of the hydrophobic organic compound can be obtained.
FIG. 3 is a calibration curve of bisphenol A (BPA) obtained as described above, with the horizontal axis indicating the concentration and the vertical axis indicating the peak height.
As shown in FIG. 3, since the peak height increases linearly with respect to the BPA concentration, the concentration of the hydrophobic organic compound contained in the sample can be determined by measuring the peak height. all right. In addition, as shown in FIG. 2, a sharp peak can be detected for each hydrophobic organic compound by elution analysis of an aqueous solution (sample) containing a plurality of hydrophobic organic compounds. It is clear that qualitative analysis and approximate quantitative analysis of hydrophobic organic compounds can be performed at one time with a single elution analysis of an aqueous solution (sample) if the retention time and calibration curve of each hydrophobic organic compound are obtained in advance. Became.
As described above, according to this example, it has been clarified that a plurality of hydrophobic organic compounds dissolved in a trace amount in water can be sharply separated by an extremely simple method. From the results of one elution analysis, it was revealed that a qualitative analysis and an approximate quantitative analysis of a hydrophobic organic compound can be performed, which is simple and extremely excellent in operability.

  The present invention relates to a method for analyzing a hydrophobic organic compound for analyzing a hydrophobic organic compound such as an endocrine disrupting substance in an aqueous solution and a column for liquid chromatography, and a permutation of the solubility of the hydrophobic organic compound in an aqueous solution and an eluent. By utilizing the fact that the elution volume varies depending on the type, hydrophobic organic compounds can be clearly separated, tailing is difficult to occur, the resolution is excellent, qualitative analysis and approximate quantitative analysis of hydrophobic organic compounds are possible, and more detailed analysis conditions It is possible to provide a method for analyzing a hydrophobic organic compound that is easy to obtain reproducible analysis results without any prior examination, and is easy to handle. It is possible to separate hydrophobic organic compounds by using the difference in solubility, so that tailing is difficult to occur and the separation performance is excellent. Wide, it can provide a remarkably excellent column for liquid chromatography good analysis results are easily operability obtain reproducible without any prior study detailed analytical conditions.

Configuration diagram of a hydrophobic organic compound analyzer using the liquid chromatograph column in Embodiment 1 Elution curves analyzed using the liquid chromatograph columns of Example 1 and Comparative Example 1 Calibration curve for bisphenol A (BPA)

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Analyzer 2 Container 3 Eluent 4,9 Liquid flow path 5 Pump 6 Sample injection part 7 Liquid chromatograph column 8 Column thermostat 10 Detector

Claims (4)

Hydrophobic column having a hydrophilic polymer having a hydrophobic polymer chain bonded to the terminal and a crosslinked polymer fine particle having a hydrophilic unsaturated monomer cross-linked with the hydrophilic compound as a filler. A separation step of passing an aqueous solution containing an organic compound and an eluent;
A detection step of detecting the hydrophobic organic compound eluted in the eluent that has passed through the column;
And a method for analyzing a hydrophobic organic compound.   The method for analyzing a hydrophobic organic compound according to claim 1, wherein the solubility of the hydrophobic organic compound in the eluent is greater than the solubility in the aqueous solution.   3. The hydrophobic organic compound according to claim 1, wherein the hydrophobic organic compound is one or more of phenol and a phenol derivative, an aromatic hydrocarbon compound, an organic acid, a nitrile compound, and an endocrine disrupting substance. Method for analyzing hydrophobic organic compounds.   Filled with fine particles of a crosslinked polymer having a hydrophilic compound having a hydrophobic polymer chain bonded to a terminal and a hydrophilic unsaturated monomer cross-linked with the hydrophilic compound. Column for liquid chromatography.

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