JP7306090B2 - Separation method of aromatic compound and supercritical fluid chromatograph - Google Patents

Description

The present invention relates to a method of separating aromatic compounds and a supercritical fluid chromatograph used to carry out the method.

Gas chromatographs are conventionally used to analyze unknown aromatic compounds contained in natural products (eg, Patent Document 1). In the analysis of aromatic compounds using gas chromatography, a sample taken from a natural product is vaporized, placed in a carrier gas flow, introduced into a column, and separated from other compounds. The separated compound is structurally analyzed, for example, by measurement with a nuclear magnetic resonance (NMR) apparatus.

However, since capillary columns generally used in gas chromatographs have a stationary phase coated on the inside of a narrow tube, the amount of compounds that can be adsorbed on the stationary phase in the capillary column is small. In NMR, at least several μg of sample is required for one measurement, but the amount of compounds that can be separated by gas chromatograph at one time is several ng. However, it is not possible to obtain the required amount of the compound for structural analysis by

Therefore, in recent years, an analysis using supercritical fluid chromatography has been attempted. Supercritical fluid chromatography uses a supercritical fluid such as carbon dioxide as a mobile phase, and separates compounds by introducing the mobile phase into which a sample is injected into a packed column packed with a packing material containing a stationary phase. Supercritical fluid chromatography uses a supercritical fluid, which has low viscosity and high diffusivity, as the mobile phase. can be separated.

In a supercritical fluid chromatograph, since a supercritical fluid is used as a mobile phase, the internal channel must be maintained at a high pressure. Therefore, the column is required to have pressure resistance and low swelling property against supercritical fluid. Therefore, silica gel-based columns that satisfy these requirements are widely used in supercritical fluid chromatography. The term "silica gel-based column" as used herein refers to a column in which the surface of silica gel is chemically modified as a stationary phase with a functional group corresponding to the properties of the target compound and packed as a packing material. For example, Patent Document 2 describes a silica gel-based column in which octadecyl groups or the like are used as a stationary phase and silica gel is chemically modified.

JP 2017-40536 A JP 2015-215320 A JP 2018-189441 A JP 2018-189449 A

Patent Document 2 shows an example of separating vitamins using supercritical fluid chromatography using a silica gel-based column. However, when an attempt was made to separate aromatic compounds using supercritical fluid chromatography using a silica gel-based column, it was found that they could not be sufficiently separated.

The problem to be solved by the present invention is to provide a technique for separating fragrant compounds using supercritical fluid chromatography.

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

In addition, the supercritical fluid chromatograph according to the present invention, which was made to solve the above problems,
an aromatic compound database containing measurement data of a plurality of aromatic compounds obtained by a predetermined measurement method;
a column packed with a packing made of a polymer having an unsaturated hydrocarbon structure;
a supercritical fluid feeder for feeding a supercritical fluid of a predetermined substance to the column;
a sample injection unit provided in a flow path from the supercritical fluid supply unit to the column for injecting a sample into the supercritical fluid;
a measurement unit that sequentially measures the effluent from the column by the predetermined measurement method;
an aromatic compound identification unit that identifies the aromatic compounds contained in the outflow by collating the measurement data obtained by the measurement with the measurement data recorded in the aromatic compound database .

The fragrant compound can be defined as a compound having a linear or cyclic unsaturated hydrocarbon structure and a molecular weight of 800 or less. Fragrant compounds have a linear or cyclic unsaturated hydrocarbon structure and thus have low polarity. Most of them are low-molecular weight compounds with a molecular weight of 800 or less, and thus have weak intermolecular bonds and are volatile. Therefore, it can also be defined as a volatile low-molecular-weight compound.
Also, the unsaturated hydrocarbon structure may be either chain or cyclic, or may contain both of them.

In the method for separating aromatic compounds and the supercritical fluid chromatograph according to the present invention, aromatic compounds are separated using a column packed with a filler made of a polymer having an unsaturated hydrocarbon structure. The reason why the silica gel-based column could not separate the aromatic compounds is that the aromatic compounds, which are low-molecular-weight compounds introduced into the column together with the highly diffusible supercritical fluid, pass through the stationary phase to the silica gel. It is believed that this is because it penetrates to the surface and causes undesirable interactions with highly polar silanol groups present on the surface. On the other hand, in the present invention, a column packed with a packing made of a polymer of a compound having an unsaturated hydrocarbon structure is used as the stationary phase. That is, the entire packing acts as a stationary phase and does not contain functional groups that cause undesired interactions, so that the fragrant compounds can be sufficiently separated.

1 is a configuration diagram of a main part of an embodiment of a supercritical fluid chromatograph according to the present invention; FIG. A chromatogram of the results of separation of aromatic compounds by a supercritical fluid chromatograph equipped with a column packed with silica gel chemically modified with an octadecyl group. A chromatogram of the results of separating aromatic compounds by a supercritical fluid chromatograph equipped with a column packed with silica gel chemically modified with a phenyl group. A chromatogram of the results of separating aromatic compounds by a supercritical fluid chromatograph equipped with a column packed with silica gel chemically modified with an aminopropyl group. 1 is a chromatogram of the result of separating an aromatic compound by carrying out one embodiment of the method for separating an aromatic compound according to the present invention using the supercritical fluid chromatograph of this example. 1 is a table summarizing the retention coefficient of each fragrant compound obtained from chromatograms obtained by the separation method of fragrant compounds of the present example and the conventional one.

An embodiment of the method for separating aromatic compounds and the supercritical fluid chromatograph (SFC ) according to the present invention will now be described with reference to the drawings.

FIG. 1 is a schematic diagram of the main part of the supercritical fluid chromatograph of this embodiment. This supercritical fluid chromatograph 1 includes a cylinder 11 containing a mobile phase substance, a pressure pump 12, a liquid container 13 containing a modifier solution to be added to the mobile phase, a pump 14, a mixer 15, a sample injection section 16, a column 17 , a back pressure control valve 18 , a spectroscopic detector 19 and a fraction collector 20 . It also has a control/processing section 4 for controlling the operation of each of these sections.

The cylinder 11 contains carbon dioxide as a mobile phase substance. Further, the liquid container 13 contains methanol as a modifier. A flow path between the pressure pump 12 and the back pressure control valve 18 is maintained at a pressure and temperature at which carbon dioxide becomes a supercritical fluid. Also, the column 17 is housed in a column oven 171 and maintained at a predetermined temperature. The column 17 of this example is packed with beads made of a polymer of styrene and divinylbenzene as a packing material. Details of the column 17 will be described later.

The spectroscopic detector 19 includes a flow cell 191 into which the sample liquid eluted from the column 17 is introduced, a lamp 192 that irradiates the sample liquid flowing through the flow cell 191 with light of a predetermined wavelength range, and the light transmitted through the flow cell 191. A light detection unit 193 is provided for separating wavelengths and measuring the intensity of light of each wavelength.

The fraction collector 20 fractionates the fragrant compound based on the detection result by the spectroscopic detector 19, and fractionates the effluent from the fractionating container 21 and the spectroscopic detector 19 into the fractionating container 21. A sorting section 22 is provided. The effluent that passes through the sorting section 22 is discarded to the drain.

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

The fragrant compound database 411 stores information including the name of the fragrant compound and the absorption spectrum previously obtained by the spectroscopic detector 19 for each of a plurality of known fragrant compounds. The measurement conditions (type and temperature of column, type and flow rate of mobile phase, etc.) for measuring aromatic compounds are also stored.

When the user instructs to start measuring fragrant compounds, the measurement control unit 421 causes the display unit 6 to display a screen for the user to select a measurement target from fragrant compounds stored in the fragrant compound database 411. indicate. When the user determines the fragrant compound to be measured by an appropriate operation, the measurement control unit 421 reads the absorption spectrum information and the measurement conditions from the fragrant compound database 411 .

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

Also, the pump 14 feeds the methanol contained in the liquid container 13 at a predetermined flow rate. Carbon dioxide and methanol are mixed by mixer 15 . In the sample injection section 16, the sample is injected into the mobile phase flow formed by mixing carbon dioxide and methanol. The sample in this example contains multiple kinds of fragrant compounds.

The sample injected into the mobile phase is introduced into the column 17 along with the flow of the mobile phase. Within column 17 , each compound in the sample interacts with the stationary phase within column 17 . Then, each compound is temporally separated according to the degree of interaction with the stationary phase and flows out from the column 17 .

Each compound discharged from the column 17 passes through the back pressure control valve 18 and is introduced into the spectroscopic detector 19 in sequence. In the spectroscopic detector 19, the flow cell 191 is irradiated with light in a predetermined range of wavelengths from the lamp 192 from the start of measurement to the end of the measurement. is measured at predetermined time intervals. The light intensity of each wavelength measured by the photodetector 193 is sequentially stored in the storage 41 .

The fragrant compound identification unit 422 creates an absorption spectrum each time measurement data is stored in the storage unit 41 and compares it with the absorption spectrum of the fragrant compound to be measured read from the fragrant compound database 411 . When both match with a predetermined accuracy or more, the measurement control unit 421 outputs a control signal to fractionate the fragrant compound to the fraction collector 20 . The fragrant compound identification unit 422 also sequentially determines whether or not a significant difference has appeared in the absorption spectra. Although the absorption spectra stored in the fragrant compound database 411 do not match, if there is a significant difference in the absorption spectra, there is a possibility that an unknown fragrant compound has flowed out of column 17 . Therefore, the measurement control unit 421 outputs a control signal to the fraction collector 20 to fractionate the effluent from the column 17 even when it is determined that a significant difference has appeared in the absorption spectra.

When a control signal instructing fractionation is output from the measurement control unit 421 , the fraction collector 20 collects the fragrant compound (or an effluent that may contain an unknown fragrant compound) into the fractionation unit 22 . They are sorted into the sorting container 21 at the timing of reaching . After completing the sorting of one odorant compound or effluent, the sorting section 22 moves downstream to the adjacent sorting container 21 and waits for a control signal from the measurement control section 421 . In this way, the desired fragrant compound and the unknown fragrant compound are sequentially fractionated into the fractionation container 21 . The effluent that flows out from the flow cell 191 of the spectroscopic detector 19 and has passed through the sorting section 22 is discarded to the drain. Here, we explained the case where both the fragrant compound to be measured and the unknown fragrant compound (the effluent that may be) are fractionated, but depending on the purpose of measurement, only one of them can be fractionated. You may do so.

Here, the column 17 used in the method for fractionating aromatic compounds and the supercritical fluid chromatograph of this example will be described.

In silica gel-based columns, which are widely used in supercritical fluid chromatography , highly polar silanol groups are present on the surface of silica gel chemically modified with functional groups. When an aromatic compound, which is a low-molecular-weight compound, is introduced together with a highly diffusible supercritical fluid, it passes through the stationary phase and penetrates to the surface of silica gel, causing undesirable interactions with silanol groups present on the surface. occur. It is speculated that these undesired interactions are the reason why the traditional inability to separate aromatic compounds on silica gel-based columns.

Since aromatic compounds generally have a linear or cyclic unsaturated hydrocarbon structure, it is effective to use a compound having an unsaturated hydrocarbon structure as the stationary phase. In Patent Documents 3 and 4, the present inventors have proposed a column filled with polymer beads having an unsaturated hydrocarbon structure (hereinafter referred to as "polymer beads"), and this column is used to separate aromatic compounds. thought to be effective for That is, when polymer beads are used as a packing material, undesirable interactions are less likely to occur between the aromatic compound to be separated and the packing material, and sharper than when silica gel or chemically modified silica is used. I thought I could get a nice peak.

Materials for the polymer beads used as the packing material for the column 17 include acrylic polymers, styrene polymers, polyacrylamide polymers, cellulose polymers, and the like. In a supercritical fluid chromatograph using supercritical fluid carbon dioxide as a mobile phase, pressure exceeding the critical pressure of carbon dioxide (7.4 MPa) is applied to the column 17 arranged in the analysis channel. is required to withstand pressure. In addition, polymer beads are required to have high solvent resistance so that they do not swell or shrink even when the mixing ratio of carbon dioxide and modifier is changed. Therefore, as the polymer beads, those having a small degree of swelling with respect to the solvent are preferably used.

Polymer beads used as column packing preferably contain crosslinked polymers. The polymer beads preferably have a degree of swelling of 1.4 or less when absorbing tetrahydrofuran and when absorbing methanol. By using such low-swelling polymer beads, a good peak shape can be obtained, and since the polymer beads have high durability, good analysis results tend to be obtained even when repeated analysis is performed. In addition, since polymer beads with a low degree of swelling can sufficiently increase the filling pressure when filling a column, deterioration of analysis performance by supercritical fluid chromatography can be suppressed. Incidentally, tetrahydrofuran is a substance that easily swells the resin material, among the substances used as the mobile phase. Methanol is also a substance that is widely used as a modifier. Therefore, by using polymeric beads as fillers that swell less for both of these, it is possible to perform measurements with a variety of mobile phases (including those with added modifiers).

The degree of swelling of polymer beads is determined based on the volume change before and after dispersing the polymer beads in a solvent. The degree of swelling of the polymer beads upon absorption of tetrahydrofuran and the degree of swelling upon absorption of methanol are more preferably 1.3 or less, more preferably 1.2 or less. The degree of swelling is generally 1.0 or more.

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

From the viewpoint of increasing the number of theoretical plates of the column, it is preferable that the coefficient of variation (CV), which is an index indicating the dispersibility of the particle size (diameter) of the polymer beads, is small, for example, 25% or less, It may be 20% or less, 15% or less or 10% or less. Although the lower limit of the CV value is not particularly limited, it is generally 1% or more. For the purpose of adjusting the average particle size and CV value, the polymer beads may be classified using an arbitrary sieve or the like.

The higher the degree of cross-linking of the polymer, the less swelling the polymer beads tend to have. The degree of cross-linking of the cross-linked polymer contained in the polymer beads is, for example, 50% or more, 80% or more, or 90% or more. If the degree of cross-linking is within the above range, the effect of the supercritical fluid is reduced, and the analysis performance can be improved. The degree of cross-linking of a cross-linked polymer is the mixing ratio of the cross-linking monomer in the monomers used for polymerization, and is defined as the mass ratio of the cross-linking monomer based on the total mass of the polymerizable monomers.

Crosslinking monomers are compounds having two or more polymerizable functional groups, and include divinyl compounds such as divinylbenzene, divinylbiphenyl, and divinylnaphthalene; diallyl phthalate and its isomers; triallyl isocyanurate and its derivatives; ) acrylic acid esters and the like. The cross-linking monomers may be used singly or in combination of two or more. Polyfunctional (meth)acrylic acid esters include di(meth)acrylic acid esters, tri- or higher functional (meth)acrylic acid esters, and the like.

Di(meth)acrylic acid esters include alkanediol di(meth)acrylates in which two (meth)acrylates are bonded to an alkylene group. The number of carbon atoms in the alkylene group may be, for example, 1-20 or 1-5. Alkylene groups may be linear, branched or cyclic. The alkylene group may have a substituent such as a hydroxyl group.

Examples of alkanediol di(meth)acrylates 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 di(meth)acrylic acid esters are ethoxylated bisphenol A based di(meth)acrylates, propoxylated ethoxylated bisphenol A based di(meth)acrylates, tricyclodecanedimethanol di(meth)acrylate, 1, Di(meth)acrylates such as 1,1-trishydroxymethylethane di(meth)acrylate, ethoxylated cyclohexanedimethanol di(meth)acrylate; and (poly)ethylene glycol di(meth)acrylate, (poly)propylene glycol di(meth)acrylate. (Meth)acrylates, (poly)alkylene glycol-based di(meth)acrylates such as (poly)tetramethylene glycol di(meth)acrylates are included.

Tri- or more functional (meth)acrylates include trimethylolpropane tri(meth)acrylate, tetramethylolmethane tri(meth)acrylate, tetramethylolpropane tetra(meth)acrylate, pentaerythritol tri(meth)acrylate, 1,1, 1-trishydroxymethylethane tri(meth)acrylate, 1,1,1-trishydroxymethylpropane triacrylate and the like.

Among these cross-linking monomers, a polymer with a high cross-linking density (a polymer with a high ratio of structures derived from polyfunctional monomers) can be easily obtained, and the degree of swelling of polymer beads can be reduced. One or more selected from the group consisting of esters may be used. That is, the crosslinked polymer may contain a structural unit derived from divinylbenzene and/or a structural unit derived from di(meth)acrylic acid ester.

Monofunctional monomers may be used along with the cross-linking monomers. Examples of monofunctional monomers include methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, isobutyl acrylate, hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, and dodecyl acrylate. , lauryl acrylate, stearyl acrylate, 2-chloroethyl acrylate, phenyl acrylate, α-methyl chloroacrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, methacrylic acid Monofunctional (meth)acrylates such as hexyl, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, and stearyl methacrylate; styrene, o-methylstyrene, m-methylstyrene, p -methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4-dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexyl Styrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecylstyrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, 3,4-dichloro Styrene such as styrene and derivatives thereof; Vinyl esters such as vinyl acetate, vinyl propionate, vinyl benzoate and vinyl butyrate; N-vinyls such as N-vinylpyrrole, N-vinylcarbazole, N-vinylindole and N-vinylpyrrolidone compounds; fluorinated monomers such as vinyl fluoride, vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, trifluoroethyl acrylate and tetrafluoropropyl acrylate; and conjugated dienes such as butadiene and isoprene. These may be used singly or in combination of two or more.

The polymer bead may consist entirely of a crosslinked polymer, or may partially contain a crosslinked polymer. From the viewpoint of lowering the degree of swelling, it is preferable that at least the outer layer of the polymer bead contains a crosslinked polymer. A polymer bead having a crosslinked polymer in its outer layer is obtained, for example, by a seed polymerization method.

In general, the smaller the particle size of the beads, the larger the theoretical plate number of the column. In general, it is sometimes difficult to form particles with a small particle size with polymer beads compared to silica gel or the like, but the seed polymerization method easily forms particles with a small particle size.

The seed polymerization method is a method in which seed particles are swollen in an emulsified liquid containing a polymerizable monomer, the seed particles absorb the polymerizable monomer, and then the polymerizable monomer is polymerized. Examples of seed particles include (meth)acrylate particles and styrene particles.

(Meth)acrylate particles are obtained by polymerization of (meth)acrylic acid ester. (Meth)acrylic acid esters include (meth)acrylic acid esters having a linear or branched alkylene group as exemplified above. Styrenic particles can be obtained, for example, by polymerization of styrenic monomers such as styrene, p-methylstyrene, p-chlorostyrene, chloromethylstyrene and α-methylstyrene. As a monomer for obtaining seed particles, allyl alcohol, allyl phthalate, allyl ether, etc. may be combined in addition to the above (meth)acrylic acid ester and styrene-based monomer. These monomers may be used singly or in combination of two or more.

The seed particles can be synthesized by a known method such as an emulsion polymerization method, a soap-free emulsion polymerization method, a dispersion polymerization method, etc., using the above monomers. The average particle size of the seed particles may be adjusted according to the designed particle size of the resulting polymer beads. The average particle size 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. The average particle size of the seed particles may be, for example, 0.1 μm or more or 0.5 μm or more from the viewpoint of efficiently obtaining seed particles that are uniform and nearly spherical. From these points of view, the average particle size of the seed particles is preferably 0.1 to 2.0 μm, more preferably 0.5 to 2.0 μm, even more preferably 0.5 to 1.5 μm.

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 resulting polymer beads. Seed particles generally have a CV value of 1% or more.

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

Polymer beads are obtained by adding seed particles to an emulsified liquid containing a polymerizable monomer and an aqueous medium, allowing the seed particles to absorb the polymerizable monomer, and then polymerizing the polymerizable monomer. Emulsions can be prepared by known methods. For example, an emulsion can be obtained by adding a polymerizable monomer to an aqueous medium and dispersing it in the aqueous medium using a fine emulsifier such as a homogenizer, an ultrasonic processor, or a nanomizer. The aqueous medium includes water or a mixed medium of water and a water-soluble solvent (eg, lower alcohol). The aqueous medium may contain a surfactant. Any of anionic, cationic, nonionic and zwitterionic surfactants may be used as the surfactant.

The emulsion may contain polymerization initiators such as organic peroxides and azo compounds, if necessary. The polymerization initiator can be used, for example, in the range of 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the polymerizable monomer.

The emulsion may contain a polymeric dispersion stabilizer such as polyvinyl alcohol or polyvinylpyrrolidone in order to improve the dispersion stability of the seed particles. The polymeric dispersion stabilizer can be used, for example, in the range of 1 to 10 parts by mass with respect to 100 parts by mass of the polymerizable monomer.

The emulsion may contain water-soluble polymerization inhibitors such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble B vitamins, citric acid, and polyphenols. By containing a polymerization inhibitor, emulsion polymerization of the monomer in the emulsion can be suppressed.

The seed particles may be added directly to the emulsion, or may be added in a state in which 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 after adding the seed particles at room temperature for 1 to 24 hours. By heating the emulsion to about 30 to 50°C, absorption of the polymerizable monomer tends to be accelerated.

The mixing ratio of the polymerizable monomer to the seed particles is not particularly limited. It may be at least parts by mass. On the other hand, for example, from the viewpoint of suppressing the unique suspension polymerization of the polymerizable monomer in the aqueous medium and efficiently producing polymer beads having the desired average particle size, the mixing ratio of the polymerizable monomer is It 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 as they absorb the polymerizable monomers, whether or not the absorption of the polymerizable monomers into the seed particles is complete can be determined by confirming the expansion of the particle size of the seed particles using an optical microscope. I can judge.

Polymer beads are obtained by polymerizing the polymerizable monomer absorbed by the seed particles. Polymerization conditions are not particularly limited, and may be appropriately selected according to the type of monomer and the like. After completion of the polymerization, the polymer beads are isolated by removing the aqueous medium from the polymerization liquid by centrifugation or filtration as necessary, washing with water and a solvent, and drying.

Polymer beads may have a porous structure. For example, porous beads can be obtained by using an organic solvent that is insoluble or sparingly soluble in an aqueous medium during seed polymerization to promote phase separation.

In the present invention, sample separation and analysis can be performed in the same manner as in conventional supercritical fluid chromatographs, except that predetermined polymer beads are used as the packing material for the separation column. 1 is not limited.

The solvent used for packing the polymer beads into 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 packing the polymer beads into the column may be, for example, 10 MPa or higher or 15 MPa or higher. By increasing the filling pressure, tailing of peaks in the chromatogram is suppressed, making it easier to obtain good peak shapes. From the viewpoint of suppressing modification of the polymer beads and damage to the column, the packing pressure may be, for example, 60 MPa or less or 50 MPa or less.

Next, the conventional supercritical fluid chromatograph (equipped with a silica gel-based column) and the supercritical fluid chromatograph of this example (equipped with a column packed with polymer beads of styrene and divinylbenzene as packing materials) ) are used to separate 23 kinds of aromatic compounds.

FIG. 2 is a chromatogram of the results of separating aromatic compounds by a conventional supercritical fluid chromatograph equipped with a column packed with silica gel chemically modified with octadecyl groups, and FIG. 3 is a silica gel chemically modified with phenyl groups. Fig. 4 is a chromatogram of the results of separating aromatic compounds by a conventional supercritical fluid chromatograph equipped with a column packed with 1 is a chromatogram of the results of separating aromatic compounds by supercritical fluid chromatography. On the other hand, FIG. 5 is a chromatogram of the results of separation of aromatic compounds by the supercritical fluid chromatograph of this example. FIG. 6 is a table summarizing the retention coefficient k' of each fragrant compound obtained from the chromatograms of FIGS.

In FIG. 2 (filled with silica gel chemically modified with octadecyl groups), 23 kinds of aromatic compounds all flowed out during the retention time period of 1.0 to 2.0 minutes, and were hardly separated. In Fig. 3 (filled with silica gel chemically modified with phenyl groups), although the fragrant compounds are separated compared to Fig. 2, all fragrant compounds are still present in the retention time range of 1.0 to 4.0 minutes. is flowing out, and the separation is still not sufficient. In FIG. 4 (filled with silica gel chemically modified with aminopropyl groups), the aromatic compounds are separated compared to FIGS. The aromatic compounds are agglomerated, 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 this example, each fragrant compound is separated, as can be clearly seen from the comparison with FIGS. Although some peaks of odorant compounds overlap for some time, the number is small and can be separated by changing the modifier or performing another chromatography after fractionation.

The embodiment described above is an example, and can be appropriately modified in accordance with the spirit of the present invention.
In the above examples, in order to fractionate the fragrant compound, it was configured to include a spectroscopic detector that detects the fragrant compound in a non-destructive manner. A configuration including a mass spectrometer instead of (or in addition to) the spectroscopic detector can also be used. Further, the fragrant compounds whose peaks partially overlap in the chromatograph shown in FIG. 5 may be collected by the fraction collector 20 and then further separated by another chromatograph.

[Aspect]
It will be appreciated by those skilled in the art that the multiple exemplary embodiments described above are specific examples of the following aspects.

(First aspect)
In the method for separating an aromatic compound according to the first aspect of the present invention, a sample containing an aromatic compound is placed in a flow of a mobile phase that is a supercritical fluid of a predetermined substance, and a polymer having an unsaturated hydrocarbon structure is separated from the polymer. introducing a column loaded with a packing material of

In the method for separating fragrant compounds according to the first aspect of the present invention, fragrant compounds are separated using a column filled with a filler made of a polymer having an unsaturated hydrocarbon structure. Highly polar silanol groups are present on the surface of silica gel that is chemically modified with functional groups in conventionally used columns. The reason why the silica gel-based column could not separate the aromatic compounds is that the aromatic compounds, which are low-molecular-weight compounds introduced into the column together with the highly diffusible supercritical fluid, pass through the stationary phase to the silica gel. It is believed that this is because it penetrates to the surface and undesirably interacts with silanol groups present on the surface. In contrast, in the first aspect of the present invention, a column packed with a packing material made of a polymer of a compound having an unsaturated hydrocarbon structure is used as the stationary phase. That is, the bulk of the packing acts as a stationary phase and is free of functional groups that cause unwanted interactions, thus allowing the separation of aromatic compounds.

(Second aspect)
A method for separating an aromatic compound according to a second aspect of the present invention is the method for separating an aromatic compound according to the first aspect,
The mobile phase is carbon dioxide in a supercritical state, or a modifier added to carbon dioxide in a supercritical state.

In the method for separating aromatic compounds according to the second aspect of the present invention, the mobile phase is mainly composed of carbon dioxide in a supercritical state. Carbon dioxide has a critical pressure of 7.38 MPa and a critical temperature of 31.1°C, and its critical temperature is close to room temperature, and it is non-flammable and chemically reactive, so it can be safely measured. In addition, high-purity products can be obtained at low cost.

(Third aspect)
A method for separating an aromatic compound according to a third aspect of the present invention is the method for separating an aromatic compound according to the first aspect or the second aspect,
The filler is a polymer bead,
The polymer beads have a degree of swelling of 1.4 or less when absorbing tetrahydrofuran and a degree of swelling when absorbing methanol,
The polymeric beads comprise crosslinked polymers.

Tetrahydrofuran, among substances used as a mobile phase, is a substance that particularly tends to swell resin materials. Methanol is also a substance that is widely used as a modifier. In the method for separating aromatic compounds according to the third aspect of the present invention, a column packed with polymer beads having a low degree of swelling in both tetrahydrofuran and methanol is used as a packing material. including ). Also, the bonds in crosslinked polymers are stronger than in non-crosslinked polymers. By using polymer beads containing such a crosslinked polymer, the degree of swelling can be reduced and pressure resistance can be ensured.

(Fourth mode)
A method for separating an aromatic compound according to a fourth aspect of the present invention is the method for separating an aromatic compound according to the third aspect,
The crosslinked polymer has one or more selected from the group consisting of structural units derived from divinylbenzene and structural units derived from di(meth)acrylic acid esters.

In the method for separating an aromatic compound according to the fourth aspect of the present invention, a crosslinked polymer having at least one selected from the group consisting of a structural unit derived from divinylbenzene and a structural unit derived from a di(meth)acrylic acid ester is used. By using it, a polymer with a high cross-linking density (a polymer with a high ratio of structures derived from polyfunctional monomers) can be easily obtained, and the degree of swelling of polymer beads can be reduced.

(Fifth aspect)
A method for separating an aromatic compound according to a fifth aspect of the present invention is the method for separating an aromatic compound according to the third aspect or the fourth aspect,
The degree of cross-linking of the cross-linked polymer is 50% or more.

As described above, the use of a crosslinked polymer tends to reduce the degree of swelling, and the higher the degree of crosslinking of the polymer, the lower the degree of swelling of the polymer beads. In the method for separating aromatic compounds according to the fifth aspect of the present invention, the degree of swelling can be reduced by using a crosslinked polymer having a degree of crosslinking of 50% or more.

(Sixth aspect)
A method for separating an aromatic compound according to a sixth aspect of the present invention is the method for separating an aromatic compound according to any one of the first to fifth aspects,
The average particle size of the polymer beads is 1-10 μm.

In the method for separating aromatic compounds according to the sixth aspect of the present invention, by setting the average particle size of the polymer beads to 10 μm or less, the opportunity for interaction between the aromatic compound and the stationary phase is increased, and the number of theoretical plates of the column is increased. can increase Also, by setting the average particle size of the polymer beads to 1 μm or more, it is possible to suppress an excessive increase in the pressure inside the column during use.

(Seventh aspect)
In the fragrant compound analysis method according to the seventh aspect of the present invention, the fragrant compound separated by the fragrant compound separation method of the first to sixth aspects is further subjected to chromatography and / or mass spectrometry. analyse.

When chromatography is performed in the aromatic compound analysis method according to the seventh aspect of the present invention, even if the separation in the aromatic compound separation methods of the first to sixth aspects is not complete, the separation can be made more complete. can do. Further, more detailed information such as the molecular structure of the fragrant compound can be obtained by performing mass spectrometry.

(Eighth aspect)
The supercritical fluid chromatograph according to the eighth aspect of the present invention is
an aromatic compound database containing measurement data of a plurality of aromatic compounds obtained by a predetermined measurement method;
a column packed with a packing made of a polymer having an unsaturated hydrocarbon structure;
a supercritical fluid feeder for feeding a supercritical fluid of a predetermined substance to the column;
a sample injection unit provided in a flow path from the supercritical fluid supply unit to the column for injecting a sample into the supercritical fluid;
a measurement unit that sequentially measures the effluent from the column by the predetermined measurement method;
an aromatic compound identification unit that identifies the aromatic compounds contained in the outflow by collating the measurement data obtained by the measurement with the measurement data recorded in the aromatic compound database .

By using the supercritical fluid chromatograph according to the eighth aspect of the present invention, aromatic compounds can be sufficiently separated as in the first aspect. In addition, by identifying the compound after separation, it is possible to easily grasp the possibility that an unknown aromatic compound is contained in the sample.

Reference Signs List 1 Supercritical fluid chromatograph 11 Cylinder 12 Pressure pump 13 Liquid container 14 Pump 15 Mixer 16 Sample injection part 17 Column 171 Column oven 18 Back pressure control valve 19 Spectral detector 191 Flow cell 192... Lamp 193... Photodetector 20... Fraction collector 21... Fractionation container 22... Fractionation part 4... Control/processing part 41... Storage part 411... Fragrant compound database 421... Measurement control part 422... Fragrant compound identification Part 5... Input part 6... Display part

Claims (8)

A sample containing a plurality of aromatic compounds is placed in a flow of a mobile phase, which is a supercritical fluid of a predetermined substance, and introduced into a column packed with a packing material made of a polymer having an unsaturated hydrocarbon structure. , a method for the separation of aromatic compounds. 2. The method for separating aromatic compounds according to claim 1, wherein the mobile phase is carbon dioxide in a supercritical state, or carbon dioxide in a supercritical state to which a modifier is added. The filler is a polymer bead,
The polymer beads have a degree of swelling of 1.4 or less when absorbing tetrahydrofuran and a degree of swelling when absorbing methanol,
3. The method for separating fragrant compounds according to claim 1 or 2, wherein the polymer beads comprise a crosslinked polymer. The method for separating fragrant compounds according to claim 3, wherein the crosslinked polymer has structural units derived from divinylbenzene. 5. The method for separating fragrant compounds according to claim 3 or 4, wherein the degree of cross-linking of said cross-linked polymer is 50% or more. The method for separating aromatic compounds according to any one of claims 3 to 5, wherein the polymer beads have an average particle size of 1 to 10 µm. A method for analyzing aromatic compounds, comprising analyzing the aromatic compounds separated into components by the method according to any one of claims 1 to 6 by chromatography and/or mass spectrometry. an aromatic compound database containing measurement data of a plurality of aromatic compounds obtained by a predetermined measurement method using supercritical fluid chromatography;
a column packed with a packing made of a polymer having an unsaturated hydrocarbon structure;
a supercritical fluid feeder for feeding a supercritical fluid of a predetermined substance to the column;
a sample injection unit provided in a flow path from the supercritical fluid supply unit to the column for injecting a sample into the supercritical fluid;
a measurement unit that sequentially measures the effluent from the column by the predetermined measurement method;
a fragrant compound identification unit that identifies a plurality of fragrant compounds contained in the outflow by collating measurement data obtained by the measurement with measurement data recorded in the fragrant compound database; Critical fluid chromatograph.

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