CN112098528A

CN112098528A - Method for separating aromatic compound and supercritical fluid chromatograph

Info

Publication number: CN112098528A
Application number: CN202010072189.0A
Authority: CN
Inventors: 藤户由佳; 马场健史
Original assignee: Shimadzu Corp
Current assignee: Shimadzu Corp
Priority date: 2019-06-17
Filing date: 2020-01-21
Publication date: 2020-12-18
Anticipated expiration: 2040-01-21
Also published as: JP2020204514A; CN112098528B; JP7306090B2

Abstract

The present invention provides a technique for separating an aromatic compound using a supercritical fluid chromatograph. The method for separating an aromatic compound comprises the following steps: a sample containing an aromatic compound is placed in a fluid flow of a mobile phase which is a supercritical fluid of a predetermined substance, and introduced into a column packed with a packing material composed of a polymer having an unsaturated hydrocarbon structure.

Description

Method for separating aromatic compound and supercritical fluid chromatograph

Technical Field

The present invention relates to a method for separating aromatic compounds and a supercritical fluid chromatograph for carrying out the method.

Background

Conventionally, a gas chromatograph has been used to analyze unknown aroma compounds contained in natural substances (for example, patent document 1). In the analysis of aromatic compounds using a gas chromatograph, a sample extracted from a natural substance is vaporized, and a gas stream carried on a carrier gas is introduced into a column to be separated from other compounds. The structure of the isolated compound can be analyzed by measuring it using a Nuclear Magnetic Resonance (NMR) apparatus, for example.

However, since a capillary column used in a gas chromatograph generally coats a stationary phase in a capillary tube, the amount of a compound that can be adsorbed to the stationary phase in the capillary column is small. In NMR, at least several μ g of a sample is required for one measurement, but the amount of a compound that can be separated at one time in a gas chromatograph is several ng, and therefore, when a gas chromatograph is used, the amount of the compound required for structural analysis by NMR cannot be obtained unless separation is repeated several times.

Therefore, in recent years, analysis using a supercritical fluid chromatograph has been attempted. In a supercritical fluid chromatograph, a supercritical fluid such as carbon dioxide is used as a mobile phase, and the mobile phase into which a sample is injected is introduced into a packed column packed with a packing material containing a stationary phase to separate a compound. In the supercritical fluid chromatography, since a supercritical fluid having low viscosity and high diffusibility is used as a mobile phase, many compounds can be separated at once by a packed column while maintaining the same advantages as those of a gas chromatograph such as high flow rate and high resolution.

In the supercritical fluid chromatography, since the supercritical fluid is used as a mobile phase, it is necessary to maintain the internal flow path at a high pressure. Therefore, the column is required to have low pressure resistance and low swelling property with respect to the supercritical fluid. Thus, silica gel-based chromatography columns satisfying these elements are widely used in supercritical fluid chromatography. The silica gel-based column is a column packed with a functional group of a type corresponding to the characteristics of the target compound as a filler, the functional group being chemically modified with respect to the surface of silica gel. For example, patent document 2 describes a column using octadecyl or the like as a matrix for immobilizing silica gel (ODS) that is chemically modified with respect to silica gel.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2017-40536

Patent document 2: japanese laid-open patent publication No. 2015-215320

Patent document 3: japanese patent laid-open publication No. 2018-189441

Patent document 4: japanese patent laid-open publication No. 2018-189449

Disclosure of Invention

Technical problem to be solved by the invention

Patent document 2 shows an example of separating vitamins by using a supercritical fluid chromatograph having a silica gel-based column. However, it was found that when separation of an aromatic compound is attempted by a supercritical fluid chromatography using a silica gel-based column, sufficient separation cannot be performed.

The technical problem to be solved by the present invention is to provide a technique for separating an aromatic compound using a supercritical fluid chromatograph.

Solution for solving the above technical problem

The method for separating an aromatic compound according to the present invention, which has been accomplished to solve the above-mentioned problems, comprises the steps of: a sample containing an aromatic compound is placed in a fluid flow of a mobile phase which is a supercritical fluid of a predetermined substance, and introduced into a column packed with a packing material composed of a polymer having an unsaturated hydrocarbon structure.

In order to solve the above-described problems, a supercritical fluid chromatograph according to the present invention includes:

An aroma compound database that records measurement data of a plurality of aroma compounds obtained by a predetermined measurement method;

a column packed with a packing material composed of a polymer having an unsaturated hydrocarbon structure;

a supercritical fluid supply unit configured to supply a supercritical fluid of a predetermined substance to the column;

a sample injection section provided in a flow path from the supercritical fluid supply section to the column, for injecting a sample into the supercritical fluid;

a measuring unit for sequentially measuring the effluents from the columns by the predetermined measuring method;

and an aromatic compound identification unit for identifying the aromatic compound contained in the effluent by comparing the measurement data obtained by the measurement with the measurement data recorded in the database of the aromatic compound.

Effects of the invention

The aromatic compound may be defined as, for example, a compound having a chain or cyclic unsaturated hydrocarbon structure and a molecular weight of 800 or less. The aromatic compound has a chain or cyclic unsaturated hydrocarbon structure and therefore has a low polarity, and since most of the aromatic compound is a low-molecular compound having a molecular weight of 800 or less, intermolecular bonding is weak and volatility is exhibited. Therefore, it can also be defined as a volatile low-molecular compound.

The unsaturated hydrocarbon structure may be either a chain or a ring, or both of them.

In the method for separating an aromatic compound and the supercritical fluid chromatograph according to the present invention, the aromatic compound is separated using a column packed with a packing material made of a polymer having an unsaturated hydrocarbon structure. It is considered that the reason why the separation of the aroma compound by the silica gel-based column is impossible is that the aroma compound, which is a low-molecular compound introduced into the column together with the supercritical fluid having high diffusibility, enters the surface of the silica gel through the stationary phase, and an undesirable interaction occurs between the aroma compound and the high-polarity silanol group present on the surface. In contrast, in the present invention, a column packed with a packing material composed of a polymer of a compound having an unsaturated hydrocarbon structure as a stationary phase is used. That is, since the entire filler acts as a stationary phase and does not contain a functional group that causes an undesired interaction, the aromatic compound can be sufficiently separated.

Drawings

Fig. 1 is a main part configuration diagram of an embodiment of a supercritical fluid chromatograph according to the present invention.

Fig. 2 is a chromatogram showing the results of separation of an aromatic compound by a supercritical fluid chromatograph equipped with a column using silica gel chemically modified with octadecyl groups as a filler.

Fig. 3 is a chromatogram showing the results of separation of an aromatic compound by a supercritical fluid chromatograph equipped with a column using silica gel chemically modified with phenyl groups as a packing material.

Fig. 4 is a chromatogram showing the results of separation of an aromatic compound by a supercritical fluid chromatograph equipped with a column using silica gel chemically modified with aminopropyl as a packing material.

Fig. 5 is a chromatogram showing the result of separation of an aromatic compound by performing an embodiment of the method for separating an aromatic compound according to the present invention with a supercritical fluid chromatograph according to the present example.

Fig. 6 is a table summarizing the retention coefficients of the aroma compounds obtained by the chromatograms obtained by the separation methods of the present example and the conventional aroma compounds.

Detailed Description

An embodiment of the method for separating an aromatic compound and a Supercritical Fluid Chromatograph (SFC) according to the present invention will be described below with reference to the drawings.

FIG. 1 is a schematic diagram showing the principal part of a supercritical fluid chromatograph according to the present embodiment. The supercritical fluid chromatograph 1 includes: a gas cylinder 11 containing a mobile phase material; a pressure pump 12; a liquid container 13 containing a modifier solution added to the mobile phase; a pump 14; a mixer 15; a sample injection section 16; a chromatography column 17; a backpressure control valve 18; a spectral detector 19 and a fraction collector 20. Further, a control processing unit 4 is provided for controlling the operations of these units.

Carbon dioxide as a mobile phase substance is contained in the gas cylinder 11. Further, methanol as a modifier is contained in the liquid container 13. The flow path between the pressurization pump 12 and the back pressure control valve 18 is maintained at the pressure and temperature at which carbon dioxide becomes a supercritical fluid. The column 17 is housed in a column oven 171 and maintained at a predetermined temperature. The column 17 of this example was packed with beads composed of a polymer (polymer) of styrene and divinylbenzene (STDVB: styrene divinyl benzene) as a packing material. Details of the column 17 will be described later.

The spectroscopic detector 19 includes: a flow cell 191 into which a sample solution eluted from the column 17 is introduced; a lamp 192 for irradiating a sample liquid flowing through the flow cell 191 with light having a wavelength in a predetermined range; and a light detection unit 193 for wavelength-separating the light transmitted through the flow cell 191 and measuring the intensity of the light of each wavelength.

The fraction collector 20 fractionates the aromatic compounds based on the detection result of the spectroscopic detector 19, and includes: a fractionation vessel 21; the fractionation section 22 fractionates the effluent from the light fractionation detector 19 into the fractionation vessel 21. The effluent passing through the fractionation unit 22 is discarded to a waste liquid flow path.

The control processing unit 4 includes a storage unit 41 of the aroma compound database 411, and further includes a measurement control unit 421 and an aroma compound identification unit 422 as functional blocks. The control processing unit 4 is a personal computer, and these functional blocks are embodied by executing an aroma compound analysis program installed in advance in the computer. The control processing unit 4 is connected to an input unit 5 including a keyboard and a mouse, and a display unit 6.

For each of the known plural aroma compounds, information including the name of the aroma compound and the absorption spectrum obtained in advance by the spectroscopic detector 19 is stored in the aroma compound database 411. In addition, measurement conditions (the type and temperature of the column, the type and flow rate of the mobile phase, and the like) for measuring the aromatic compound are also stored.

When the user instructs the start of measurement of the aromatic compounds, the measurement control unit 421 displays a screen on the display unit 6, which allows the user to select a measurement target from the aromatic compounds stored in the aromatic compound database 411. When the user determines the aroma compound to be measured by an appropriate operation, the measurement control unit 421 reads the information of the absorption spectrum and the measurement condition from the aroma compound database 411.

Next, the measurement control unit 421 heats the column 17 to a predetermined temperature based on the measurement conditions read from the aroma compound database 411. When the column 17 is heated to this temperature, the measurement control unit 421 operates the pressurization pump 12 to pressurize the carbon dioxide gas contained in the gas cylinder 11 to a predetermined pressure based on the measurement conditions and supply the carbon dioxide gas at a predetermined flow rate. Carbon dioxide is supplied in a supercritical state in the flow path between the pressurizing pump 12 and the back pressure control valve 18.

The methanol contained in the liquid container 13 is supplied at a predetermined flow rate by a pump 14. The carbon dioxide is mixed with methanol by a mixer 15. In the sample injection unit 16, the sample is injected into a fluid flow of a mobile phase in which carbon dioxide and methanol are mixed. The samples in this example contain various types of aromatic compounds.

The sample injected into the mobile phase is carried in the flow of the mobile phase and introduced into the column 17. Within the column 17, each compound in the sample interacts with a stationary phase within the column 17. Then, the respective compounds are separated over time according to the size and magnitude of the interaction with the stationary phase, and flow out of the chromatographic column 17.

Each compound flowing out of the column 17 is introduced into the spectroscopic detector 19 through the back pressure control valve 18 in order. In the spectroscopic detector 19, light having a wavelength in a predetermined range is irradiated from the lamp 192 to the flow cell 191 from the start of measurement to the end of measurement, and the intensity of the light transmitted through the flow cell 191 is measured at predetermined time intervals at different wavelengths by the light detector 193. The intensities of the light of the respective wavelengths measured by the light detector 193 are sequentially stored in the storage unit 41.

Every time the measurement data is stored in the storage unit 41, the aroma compound identification unit 422 generates an absorption spectrum, and compares the absorption spectrum with the absorption spectrum of the aroma compound to be measured read from the aroma compound database 411. When both of them match each other to a level of a predetermined accuracy or more, the measurement control unit 421 outputs a control signal for fractionating the aroma compound into the fraction collector 20. The aroma compound identification unit 422 also sequentially determines whether or not an intentional difference occurs in the absorption spectrum. In the case where the absorption spectra stored in the aroma compound database 411 are not uniform but intentionally poor, there may be an unknown aroma compound flowing out of the column 17. Then, even when it is determined that an intentional difference occurs in the absorption spectrum, the measurement control unit 421 outputs a control signal for fractionating the effluent from the column 17 into the fraction collector 20.

When a control signal indicating fractionation is output from the measurement control unit 421, the aroma compound (or the effluent which may contain an unknown aroma compound) is fractionated into the fractionation vessel 21 at the timing when the aroma compound reaches the fractionation unit 22 in the fraction collector 20. When the fractionation of one of the aromatic compounds or the effluent is completed, the fractionation unit 22 moves to a position of the fractionation vessel 21 adjacent to the downstream side, and waits for a control signal from the measurement control unit 21. Thereby, the desired aroma compound and the unknown aroma compound are sequentially fractionated into the fractionation vessel 21. The effluent flowing out of the flow cell 191 of the spectroscopic detector 19 and passing through the fractionating unit 22 is discarded to the waste liquid flow path. Here, the case of fractionating both the aroma compound to be measured and the unknown aroma compound (which may be an effluent of the unknown aroma compound) is described, but only either one may be fractionated depending on the purpose of measurement.

Here, the method for fractionating an aromatic compound and the column 17 used in the supercritical fluid chromatograph according to the present embodiment will be described.

In silica gel-based chromatography columns widely used in supercritical fluid chromatography, silanol groups of high polarity are present on the surface of silica gel chemically modified with functional groups. When an aromatic compound, which is a low-molecular compound, is introduced together with a supercritical fluid having high diffusibility, the aromatic compound enters the surface of silica gel through the stationary phase, and an undesirable interaction occurs between the aromatic compound and silanol groups present on the surface. It is presumed that the reason why the aromatic compound cannot be separated by the silica gel-based column in the past is such an undesirable interaction.

Generally, since an aromatic compound has a chain or cyclic unsaturated hydrocarbon structure, it is effective to use a compound having an unsaturated hydrocarbon structure as the stationary phase. It is considered that the present inventors proposed a column packed with beads of a polymer having an unsaturated hydrocarbon structure (hereinafter referred to as "polymer beads") in patent document 3 and patent document 4, which is also effective for separation of an aromatic compound. That is, it is considered that when polymer beads are used as the filler, undesirable interaction between the aromatic compound to be separated and the filler is less likely to occur, and a sharp peak can be obtained, as compared with the case of using silica gel or chemically modified silica.

Examples of the material of the polymer beads used as the packing material of the column 17 include acrylic polymers, styrene polymers, polyacrylamide polymers, and cellulose polymers. In a supercritical fluid chromatograph using supercritical fluid carbon dioxide as a mobile phase, a pressure exceeding the critical pressure (7.4MPa) of carbon dioxide is applied to a column 17 disposed in an analysis flow path, and therefore, it is required that polymer beads as a packing material have pressure resistance. Further, even in the case of changing the mixing ratio of carbon dioxide and the modifier, the polymer beads are required to have high solvent resistance so that swelling or shrinkage does not occur. Therefore, it is preferable to use a substance having a small degree of swelling with respect to the solvent as the polymer beads.

The polymer beads used as a packing material for a chromatography column preferably contain a crosslinked polymer. The polymer beads preferably have a swelling degree of 1.4 or less in both tetrahydrofuran absorption and methanol absorption. By using such polymer beads with a low degree of swelling, a good peak shape can be obtained, and the durability of the polymer beads is high, so that even when repeated analysis is performed, a good analysis result tends to be obtained. Further, since the low-swelling polymer beads can sufficiently increase the packing pressure when packing the column, the decrease in the analytical performance by the supercritical fluid chromatography can be suppressed. Tetrahydrofuran is a substance that particularly easily swells resin materials among substances used as a mobile phase. Further, methanol is a substance widely used as a modifier. Therefore, by using polymer beads having a low degree of swelling with respect to both of them as a filler, measurements can be performed using various mobile phases (including a mobile phase to which a modifier is added).

The degree of swelling of the polymer beads was determined based on the change in volume before and after dispersing the polymer beads into the solvent. The polymer beads have a swelling degree when absorbing tetrahydrofuran and a swelling degree when absorbing methanol of more preferably 1.3 or less, and still more preferably 1.2 or less. The degree of swelling is usually 1.0 or more.

From the viewpoint of easily obtaining a column having a high theoretical number of stages, the average particle diameter of the polymer beads may be 10 μm or less, 5 μm or less, or 4 μm or less. The average particle diameter of the polymer beads may be, for example, 1 μm or more or 2 μm or more from the viewpoint of suppressing an excessive increase in column pressure during use.

From the viewpoint of increasing the number of theoretical stages of the column, the value of the Coefficient of Variation (CV: Coefficient of Variation) which is an index showing the dispersibility of the particle diameter (diameter) of the polymer beads is preferably small, and may be, for example, 25% or less, 20% or less, 15% or less, or 10% or less. The lower limit of the CV value is not particularly limited, but is usually 1% or more. The polymer beads may be classified using an arbitrary sieve or the like for the purpose of adjusting the average particle diameter, CV value, or the like.

The higher the degree of crosslinking of the polymer, the lower the degree of swelling of the polymer beads. The degree of crosslinking of the crosslinked polymer contained in the polymer beads is, for example, 50% or more, 80% or more, or 90% or more. When the degree of crosslinking is within the above range, the influence of the supercritical fluid is less likely to occur, and the analytical performance can be improved. The crosslinking degree of the crosslinked polymer is a blending ratio of the crosslinking monomer in the monomers used for polymerization, and is defined as a mass ratio of the crosslinking monomer based on the total mass of the polymerizable monomers.

The crosslinking monomer is a compound having two or more polymerizable functional groups, and examples thereof include divinyl compounds such as divinylbenzene, divinylbiphenyl, and divinylnaphthalene; diallyl phthalate and isomers thereof; triallyl isocyanurate and derivatives thereof; polyfunctional (meth) acrylates, and the like. The crosslinking monomer may be used alone or in combination of two or more. Examples of the polyfunctional (meth) acrylate include di (meth) acrylates and tri or more functional (meth) acrylates.

Examples of di (meth) acrylates include alkylene glycol di (meth) acrylates in which two (meth) acrylates are bonded to an alkylene group. The number of carbon atoms of the alkyl group may be, for example, 1 to 20 or 1 to 5. The alkylene group may be linear, branched or cyclic. The alkylene group may have a substituent such as a hydroxyl group.

Examples of the alkanediol di (meth) acrylate include 1, 3-butanediol diacrylate, 1, 4-butanediol di (meth) acrylate, 1, 5-pentanediol di (meth) acrylate, 1, 6-hexanediol di (meth) acrylate, 1, 7-heptanediol di (meth) acrylate, 1, 8-octanediol di (meth) acrylate, 3-methyl-1, 5-pentanediol di (meth) acrylate, 1, 9-nonanediol di (meth) acrylate, 1, 10-decanediol di (meth) acrylate, neopentyl glycol di (meth) acrylate, and glycerol dimethacrylate.

Other examples of the di (meth) acrylate include di (meth) acrylates such as ethoxylated bisphenol A di (meth) acrylate, propoxylated ethoxylated bisphenol A di (meth) acrylate, tricyclodecane dimethanol di (meth) acrylate, 1, 1, 1-trimethylolethane di (meth) acrylate, and ethoxylated cyclohexane dimethanol di (meth) acrylate; and (poly) alkylene glycol di (meth) acrylates such as (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, and (poly) tetramethylene glycol di (meth) acrylate.

Examples of the trifunctional or higher-functional (meth) acrylate include trimethylolpropane tri (meth) acrylate, tetramethylolmethane tri (meth) acrylate, tetramethylolpropane tetra (meth) acrylate, pentaerythritol tri (meth) acrylate, 1, 1, 1-trimethylolethane tri (meth) acrylate, and 1, 1, 1-trimethylolpropane triacrylate.

Among these crosslinking monomers, one or more selected from the group consisting of divinylbenzene and di (meth) acrylate can be used because a polymer having a high crosslinking density (a polymer having a high proportion of structures derived from a polyfunctional monomer) can be easily obtained and the degree of swelling of the polymer beads can be reduced. That is, the crosslinked polymer may contain a structural unit derived from divinylbenzene and/or a structural unit derived from di (meth) acrylate.

It is also possible to use both crosslinking monomers and monofunctional monomers. Examples of the monofunctional monomer include monofunctional (meth) acrylates such as methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, isobutyl acrylate, hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, dodecyl acrylate, lauryl acrylate, stearyl acrylate, 2-chloroethyl acrylate, phenyl acrylate, methyl α -chloroacrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, and stearyl methacrylate; styrene and derivatives thereof such as styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α -methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2, 4-dimethylstyrene, p-n-butylstyrene, p-t-butylstyrene, p-n-hexylstyrene, p-n-octylstyrene, p-n-nonylstyrene, p-n-decylstyrene, p-n-dodecylstyrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, and 3, 4-dichlorostyrene; vinyl esters such as vinyl acetate, vinyl propionate, vinyl benzoate, and vinyl butyrate; n-vinyl compounds such as N-vinylpyrrole, N-vinylcarbazole, N-vinylindole, and N-vinylpyrrolidone; fluorine-containing monomers such as fluorinated ethylene, vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, trifluoroethyl acrylate, and tetrafluoropropyl acrylate; conjugated dienes such as butadiene and isoprene. These may be used alone or in combination of two or more.

The polymer beads may be entirely composed of a crosslinked polymer, or may have a crosslinked polymer in a part thereof. From the viewpoint of reducing the degree of swelling, it is preferable that at least the outer layer of the polymer beads contain a crosslinked polymer. The polymer beads having a crosslinked polymer in the outer layer can be obtained by, for example, seed polymerization.

Generally, the smaller the particle size of the beads, the larger the number of theoretical stages of the column tends to be. In general, polymer beads are difficult to form particles having a small particle size in comparison with silica gel and the like, but the seed polymerization method is easy to form particles having a small particle size.

The seed polymerization method is a method of swelling seed particles in an emulsion containing a polymerizable monomer, allowing the seed particles to absorb the polymerizable monomer, and then polymerizing the polymerizable monomer. Examples of the seed particles include (meth) acrylate particles and styrene particles.

The (meth) acrylate particles can be obtained by polymerization of (meth) acrylate. Examples of the (meth) acrylate include the above-exemplified (meth) acrylates having a linear or branched alkyl group. The styrene-based particles can be obtained by polymerizing styrene-based monomers such as styrene, p-methylstyrene, p-chlorostyrene, chloromethylstyrene, and α -methylstyrene. As the monomer for obtaining the seed particles, in addition to the above (meth) acrylic acid ester and styrene monomer, allyl alcohol, allyl phthalate, allyl ether, and the like may be combined. These monomers may be used alone or in combination of two or more.

The seed particles can be synthesized by a known method such as emulsion polymerization, soap-free emulsion polymerization, or dispersion polymerization using the above monomers. The average particle diameter of the seed particles may be adjusted according to the design particle diameter of the obtained polymer beads. The average particle diameter of the seed particles may be, for example, 2.0 μm or less or 1.5 μm or less from the viewpoint of shortening the absorption time of the polymerizable monomer. From the viewpoint of efficiently obtaining uniform and nearly spherical seed particles, the average particle diameter of the seed particles may be, for example, 0.1 μm or more or 0.5 μm or more. From these viewpoints, the average particle diameter of the seed particles is preferably 0.1 to 2.0. mu.m, more preferably 0.5 to 2.0. mu.m, and still more preferably 0.5 to 1.5. mu.m.

The CV value of the seed particles may be, for example, 10% or less or 7% or less from the viewpoint of sufficiently ensuring the uniformity of the obtained polymer beads. The CV value of the seed particles is usually 1% or more.

The average particle diameter of the polymer beads may be adjusted to 2 to 10 times or 2.5 to 7 times the average particle diameter of the seed particles, for example. By adjusting the average particle diameter of the polymer beads to the above range, the particle diameter of the polymer beads becomes monodispersity, and the CV value of the particle diameter can be reduced.

The polymer beads can be obtained by adding seed particles to an emulsion containing a polymerizable monomer and an aqueous medium, allowing the seed particles to absorb the polymerizable monomer, and then polymerizing the polymerizable monomer. The emulsion can be prepared by a known method. For example, an emulsion can be obtained by adding a polymerizable monomer to an aqueous medium and dispersing the resultant in the aqueous medium using a fine emulsifier such as a homogenizer, an ultrasonic processor, or a high-pressure homogenizer (Nanomizer). The aqueous medium may be water or a mixed medium of water and a water-soluble solvent (e.g., a lower alcohol). A surfactant may be included in the aqueous medium. As the surfactant, any of anionic, cationic, nonionic, and zwitterionic surfactants can be used.

The emulsion may contain a polymerization initiator such as an organic peroxide or an azo compound, if necessary. For example, the polymerization initiator may be used in an amount of 0.1 to 7.0 parts by mass per 100 parts by mass of the polymerizable monomer.

In order to improve the dispersion stability of the seed particles, the emulsion may contain a polymer dispersion stabilizer such as polyvinyl alcohol or polyvinylpyrrolidone. For example, the polymer dispersion stabilizer may be used in an amount of 1 to 10 parts by mass based on 100 parts by mass of the polymerizable monomer.

The emulsion may contain water-soluble polymerization inhibitors such as nitrites, sulfites, hydroquinone, ascorbic acids, water-soluble vitamin B compounds, citric acids, polyphenols, etc. The inclusion of the polymerization inhibitor can suppress emulsion polymerization of the monomer in the emulsion.

The seed particles may be added directly to the emulsion or may be added in a state where the seed particles are dispersed in an aqueous dispersion. For example, the seed particles can be allowed to absorb the polymerizable monomer by stirring the emulsion to which the seed particles are added at room temperature for 1 to 24 hours. The absorption of the polymerizable monomer tends to be accelerated by heating the emulsion to about 30 to 50 ℃.

The mixing ratio of the polymerizable monomer to the seed particles is not particularly limited, and may be 800 parts by mass or more or 1500 parts by mass or more per 100 parts by mass of the seed particles, for example, from the viewpoint of efficiently producing polymer beads having a desired average particle diameter. On the other hand, for example, from the viewpoint of suppressing suspension polymerization of the polymerizable monomer alone in the aqueous medium and efficiently producing polymer beads having a target average particle diameter, the mixing ratio of the polymerizable monomer may be 100000 parts by mass or less or 35000 parts by mass or less with respect to 100 parts by mass of the seed particles. Since the seed particles swell by absorbing the polymerizable monomer, it is possible to determine whether or not the absorption of the polymerizable monomer into the seed particles is completed by confirming the enlargement of the particle diameter of the seed particles using an optical microscope.

The polymer beads can be obtained by polymerizing the polymerizable monomer absorbed by the seed particles. The polymerization conditions are not particularly limited, and may be appropriately selected depending on the kind of the monomer. After completion of the polymerization, the aqueous medium is removed from the polymerization solution by centrifugation or filtration as necessary, and the polymer beads are separated by washing with water and a solvent and drying.

The polymer beads may have a porous structure. For example, in the seed polymerization, a porous bead can be obtained by promoting phase separation using an organic solvent that is insoluble or poorly soluble in an aqueous medium.

In the present invention, separation and analysis of a sample can be performed in the same manner as in a conventional supercritical fluid chromatograph except that predetermined polymer beads are used as a filler of a separation column, and the configuration of the supercritical fluid chromatograph is not limited to the configuration shown in fig. 1.

The solvent used for packing the polymer beads in the column is not particularly limited as long as it can disperse the polymer beads, and examples thereof include water, methanol, THF, acetonitrile, chloroform, ethylene glycol, and liquid paraffin. The packing pressure when the polymer beads are packed in the column may be, for example, 10MPa or more or 15MPa or more. By increasing the filling pressure, tailing of the peak in the chromatogram of the supercritical fluid chromatograph is suppressed, and a good peak shape is easily obtained. The packing pressure may be, for example, 60MPa or less or 50MPa or less from the viewpoint of suppressing alteration of the polymer beads or breakage of the column.

Next, the results of separating 23 types of aroma compounds using a conventional supercritical fluid chromatograph (a column provided with a silica gel matrix) and the supercritical fluid chromatograph of the present example (a column provided with polymer beads packed with styrene and divinylbenzene as a packing material) will be described.

Fig. 2 is a chromatogram showing the results of separation of an aromatic compound by a conventional supercritical fluid chromatograph equipped with a column using octadecyl group-chemically modified silica gel as a filler, fig. 3 is a chromatogram showing the results of separation of an aromatic compound by a conventional supercritical fluid chromatograph equipped with a column using phenyl group-chemically modified silica gel as a filler, and fig. 4 is a chromatogram showing the results of separation of an aromatic compound by a conventional supercritical fluid chromatograph equipped with a column using aminopropyl group-chemically modified silica gel as a filler. On the other hand, fig. 5 is a chromatogram showing the result of separating the aromatic compounds by the supercritical fluid chromatograph of the present example. Fig. 6 is a table summarizing the retention coefficients k' of the aroma compounds obtained from the chromatograms in fig. 2 to 5.

In fig. 2 (silica gel chemically modified with octadecyl groups was used as a filler), all of the 23 aroma compounds flowed out over a retention time of 1.0 to 2.0 minutes, and were hardly separated. In fig. 3 (silica gel chemically modified with phenyl groups as a filler), although the aromatic compounds were separated as compared with fig. 2, all of the aromatic compounds flowed out over a retention time of 1.0 to 4.0 minutes, and the separation was still insufficient. In fig. 4 (silica gel chemically modified by aminopropyl group is used as a filler), although the aromatic compounds are separated as compared with fig. 2 and 3, most of the aromatic compounds are solidified during the retention time of 1.0 to 2.5 minutes, and the separation is insufficient to fractionate the aromatic compounds.

On the other hand, in the chromatogram of fig. 5 obtained by the supercritical fluid chromatograph of the present example, it is apparent from comparison with fig. 2 to 4 that the respective aroma compounds are separated. Although the time when some peaks of the aromatic compound overlap is short, the number of peaks is small, and the separation can be performed by changing the modifier or performing another chromatography after the fractionation.

The above embodiments are examples, and can be modified as appropriate in accordance with the spirit of the present invention.

In the above-described embodiment, the spectral detector that nondestructively detects the aroma compound is provided for fractionating the aroma compound, but when fractionation of the aroma compound is not necessary, a mass analyzer may be provided instead of the spectral detector (or in addition to the spectral detector). In the chromatogram shown in fig. 5, for aromatic compounds in which some peaks overlap, these compounds may be extracted by the fraction collector 20 and then separated by another chromatograph.

[ solution ]

Those skilled in the art will appreciate that the various exemplary embodiments described above are specific examples of the following aspects.

(embodiment 1)

The method for separating an aromatic compound according to claim 1 of the present invention comprises the steps of: a sample containing an aromatic compound is placed in a fluid flow of a mobile phase which is a supercritical fluid of a predetermined substance, and introduced into a column packed with a packing material composed of a polymer having an unsaturated hydrocarbon structure.

In the method for separating an aromatic compound according to claim 1 of the present invention, the aromatic compound is separated using a column packed with a packing material composed of a polymer having an unsaturated hydrocarbon structure. In a conventionally used column, silanol groups having high polarity are present on the surface of silica gel chemically modified with functional groups. It is considered that the reason why the separation of the aroma compound by the silica gel-based column is impossible is that the aroma compound, which is a low-molecular compound introduced into the column together with the supercritical fluid having high diffusibility, enters the surface of the silica gel through the stationary phase, and an undesired interaction occurs between the aroma compound and the silanol group present on the surface. In contrast, in the present invention, a column packed with a packing material composed of a polymer of a compound having an unsaturated hydrocarbon structure as a stationary phase is used. That is, since the entire filler functions as a stationary phase and does not include a functional group that can cause an undesired interaction, the aromatic compound can be separated.

(embodiment 2)

The method for separating an aromatic compound according to claim 2 of the present invention is the method for separating an aromatic compound according to claim 1,

the mobile phase is carbon dioxide in a supercritical state or a substance obtained by adding a modifier to carbon dioxide in a supercritical state.

In the method for separating an aromatic compound according to claim 2 of the present invention, the supercritical carbon dioxide mainly constitutes a mobile phase. The critical pressure of carbon dioxide is 7.38MPa and the critical temperature is 31.1 ℃, and since the critical temperature is close to normal temperature and there is no flammability and chemical reactivity, the measurement can be safely performed. Further, high-purity carbon dioxide can be obtained at low cost.

(embodiment 3)

The method for separating an aromatic compound according to claim 3 of the present invention is the method for separating an aromatic compound according to claim 1 or 2,

the filler is a polymer bead, and the filler is a polymer bead,

the polymer beads have a swelling degree of 1.4 or less in both tetrahydrofuran absorption and methanol absorption,

the polymer beads comprise a crosslinked polymer.

Tetrahydrofuran is a substance that particularly easily swells resin materials among substances used as a mobile phase. Further, methanol is a substance widely used as a modifier. In the method for separating an aromatic compound according to claim 3 of the present invention, since a column packed with polymer beads having a low degree of swelling with respect to both tetrahydrofuran and methanol as a packing is used, measurements can be performed using various mobile phases (including a mobile phase to which a modifier is added). Furthermore, the bonding within the crosslinked polymer is stronger than within the uncrosslinked polymer. By using polymer beads containing such a crosslinked polymer, the degree of swelling can be reduced and the pressure resistance can be ensured.

(embodiment 4)

The method for separating an aromatic compound according to claim 4 of the present invention is the method for separating an aromatic compound according to claim 3,

the crosslinked polymer has one or more structural units selected from the group consisting of a structural unit derived from divinylbenzene and a structural unit derived from di (meth) acrylate.

In the method for separating an aromatic compound according to claim 4 of the present invention, by using a crosslinked polymer having at least one structural unit selected from the group consisting of a structural unit derived from divinylbenzene and a structural unit derived from di (meth) acrylate, a polymer having a high crosslinking density (a polymer having a high proportion of structures derived from polyfunctional monomers) can be easily obtained, and the degree of swelling of polymer beads can be reduced.

(embodiment 5)

The method for separating an aromatic compound according to claim 5 of the present invention is the method for separating an aromatic compound according to claim 3 or 4,

the crosslinked polymer has a degree of crosslinking of 50% or more.

As described above, by using a polymer having crosslinking, the degree of swelling becomes small, and the degree of swelling of the polymer beads tends to become small as the degree of crosslinking of the polymer becomes higher. In the method for separating an aromatic compound according to claim 5 of the present invention, the swelling degree can be reduced by using a crosslinked polymer having a crosslinking degree of 50% or more.

(embodiment 6)

The method for separating an aromatic compound according to claim 6 of the present invention is the method for separating an aromatic compound according to any one of the above-mentioned aspects 1 to 5,

the polymer beads have an average particle diameter of 1 to 10 μm.

In the method for separating an aromatic compound according to claim 6 of the present invention, the chance of interaction between the aromatic compound and the stationary phase can be increased and the number of theoretical stages of the column can be increased by setting the average particle diameter of the polymer beads to 10 μm or less. Further, by setting the average particle diameter of the polymer beads to 1 μm or more, an excessive increase in pressure in the column during use can be suppressed.

(embodiment 7)

In the method for separating an aromatic compound according to claim 7 of the present invention, the aromatic compound separated by the method for separating an aromatic compound according to any one of claims 1 to 6 is further analyzed by chromatography and/or mass analysis.

When the chromatographic analysis is performed in the method for separating an aromatic compound according to claim 7 of the present invention, even when the separation is not complete in the methods for separating an aromatic compound according to claims 1 to 6, the separation can be further completed. Further, by performing mass analysis, more detailed information such as the molecular structure of the aromatic compound can be obtained.

(claim 8)

A supercritical fluid chromatograph according to claim 8 of the present invention includes:

a measuring unit for sequentially measuring the effluent from the column by the predetermined measuring method;

When the supercritical fluid chromatograph according to claim 8 of the present invention is used, the aromatic compound can be sufficiently separated in the same manner as in claim 1. In addition, when the sample contains an unknown aromatic compound, the possibility can be easily grasped by identifying the compound after separation.

Description of the reference numerals

1 supercritical fluid chromatograph

11 gas cylinder

12 pressure pump

13 liquid container

14 pump

15 mixer

16 sample injection part

17 column chromatography

171 column oven

18 backpressure control valve

19 spectroscopic detector

191 flow cell

192 Lamp

193 light detecting part

20 fraction collector

21 fractionating container

22 fractionation section

4 control processing part

41 storage part

411 database of aromatic compounds

421 measurement control part

422 aromatic compound identification part

5 input unit

6 a display part.

Claims

1. A method for separating an aromatic compound, comprising the steps of:

a sample containing an aromatic compound is placed in a fluid flow of a mobile phase which is a supercritical fluid of a predetermined substance, and introduced into a column packed with a packing material composed of a polymer having an unsaturated hydrocarbon structure.

2. The method for separating an aromatic compound according to claim 1,

3. The method for separating an aromatic compound according to claim 1,

the filler is a polymer bead, and the filler is a polymer bead,

The polymer beads comprise a crosslinked polymer.

4. The method for separating an aromatic compound according to claim 3,

5. The method for separating an aromatic compound according to claim 3,

the crosslinked polymer has a degree of crosslinking of 50% or more.

6. The method for separating an aromatic compound according to claim 3,

the polymer beads have an average particle diameter of 1 to 10 μm.

7. A method for analyzing an aromatic compound, characterized in that,

the method of claim 1, wherein the aroma compounds separated from the composition by the method of claim 1 are analyzed by chromatography and/or mass spectrometry.

8. A supercritical fluid chromatograph, comprising: