JP2020204514A - Aromatic compound separation method and supercritical fluid chromatograph - Google Patents

Abstract

To provide technology that separates an aromatic compound using a supercritical fluid chromatograph.SOLUTION: The present invention includes a step for introducing a sample that contains an aromatic compound into a column filled with a filler composed of a polymer having an unsaturated hydrocarbon structure by moving it with a mobile-phase flow which is a supercritical fluid of a prescribed substance. The present invention comprises: an aromatic compound database in which is recorded the data of a plurality of aromatic compounds acquired by a prescribed measurement method; a column filled with a filter composed of a polymer having an unsaturated hydrocarbon structure; a supercritical fluid feeding unit for feeding the supercritical fluid of a prescribed substance to the column; a sample injection unit for injecting a sample to the supercritical fluid and provided in a passage leading from the supercritical fluid feeding unit to the column; a measurement unit for sequentially measuring effluents from the column by a prescribed measurement method; and an aromatic compound identification unit for checking the data obtained by measurement against the data recorded in the aromatic compound database and thereby identifying the aromatic compound included in the effluents.SELECTED DRAWING: Figure 1

Description

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

Conventionally, a gas chromatograph has been used to analyze an unknown aroma compound contained in a natural product (for example, Patent Document 1). In the analysis of aroma compounds using a gas chromatograph, a sample collected from a natural product is vaporized, placed on a stream of carrier gas, introduced into a column, and separated from other compounds. The separated compounds are structurally analyzed, for example, by measuring with a Nuclear Magnetic Resonance (NMR) device.

However, since the capillary column generally used in a gas chromatograph has a stationary phase coated on the inside of a capillary tube, the amount of a compound 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 since the amount of compounds that can be separated at one time by gas chromatography is several ng, NMR must be repeated many times when using gas chromatography. It is not possible to obtain the amount of compound required for structural analysis by.

Therefore, in recent years, an 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 a mobile phase in which a sample is injected into a packed column loaded with a filler containing a stationary phase is introduced to separate compounds. Since the supercritical fluid chromatograph uses a supercritical fluid with low viscosity and high diffusivity as the mobile phase, many compounds can be compounded at once by the packed column while maintaining the same advantages as the gas chromatograph of high flow velocity and high resolution. Can be separated.

In a supercritical fluid chromatograph, since a supercritical fluid is used as a mobile phase, the internal flow path must be maintained at a high pressure. Therefore, the column is required to have low pressure resistance and low swelling property with respect to a supercritical fluid. Therefore, in supercritical fluid chromatographs, silica gel-based columns that meet these requirements are widely used. The silica gel-based column refers to a column loaded with a chemical modification on the surface of silica gel with a functional group of a type corresponding to the characteristics of the target compound as a stationary phase as a filler. For example, Patent Document 2 describes a silica gel-based column in which silica gel is chemically modified with an octadecyl group or the like as a stationary phase.

JP-A-2017-40536 JP-A-2015-215320 Japanese Unexamined Patent Publication No. 2018-189441 JP-A-2018-189449

Patent Document 2 shows an example in which vitamins are separated by using a supercritical fluid chromatograph using a silica gel-based column. However, when an attempt was made to separate the aroma compound using a supercritical fluid chromatograph using a silica gel-based column, it was found that the separation was not sufficient.

An object to be solved by the present invention is to provide a technique for separating aroma compounds using a supercritical fluid chromatograph.

The method for separating an aroma compound according to the present invention, which has been made to solve the above problems, is
It comprises the step of introducing a sample containing an aroma compound into a column loaded with a filler made of a polymer having an unsaturated hydrocarbon structure by placing it in a flow of a mobile phase which is a supercritical fluid of a predetermined substance.

In addition, the supercritical fluid chromatograph according to the present invention made to solve the above problems is
An aroma compound database containing measurement data of a plurality of aroma compounds acquired by a predetermined measurement method, and
A column loaded with a filler made of a polymer having an unsaturated hydrocarbon structure,
A supercritical fluid feeding unit that feeds a supercritical fluid of a predetermined substance to the column,
A sample injection unit provided in the flow path from the supercritical fluid feeding unit to the column and injecting a sample into the supercritical fluid,
A measuring unit that sequentially measures the effluent from the column by the predetermined measuring method,
It is provided with an aroma compound identification unit that identifies the aroma compound contained in the effluent by collating the measurement data obtained by the measurement with the measurement data recorded in the database of the aroma compound.

The aroma compound can be defined as, for example, a compound having a chain-like or cyclic unsaturated hydrocarbon structure and having a molecular weight of 800 or less. Aroma compounds have a small polarity because they have a chain or cyclic unsaturated hydrocarbon structure, and most of them are low molecular weight compounds having a molecular weight of 800 or less, so that they have weak molecular bonds and are volatile. Therefore, it can also be defined as a volatile low molecular weight compound.
Further, the unsaturated hydrocarbon structure may have only one of a chain shape and a cyclic shape, or may include both of them.

In the method for separating aroma compounds and the supercritical fluid chromatograph according to the present invention, the aroma compounds are separated using a column loaded with a filler made of a polymer having an unsaturated hydrocarbon structure. The reason why the aroma compounds could not be separated by the silica gel-based column was that the aroma compounds, which are low molecular weight compounds introduced into the column together with the highly diffusible supercritical fluid, passed through the stationary phase of the silica gel. It is considered that this is because it penetrated to the surface and an undesired interaction with the highly polar silanol group existing on the surface occurred. On the other hand, in the present invention, a column loaded with a filler made of a polymer of a compound having an unsaturated hydrocarbon structure, which is a stationary phase, is used. That is, since the entire filler functions as a stationary phase and does not contain functional groups that cause undesired interactions, the aroma compounds can be sufficiently separated.

FIG. 6 is a block diagram of a main part of an embodiment of a supercritical fluid chromatograph according to the present invention. A chromatogram of the result of separating aroma compounds by a supercritical fluid chromatogram equipped with a column containing silica gel chemically modified with an octadecyl group as a filler. A chromatogram of the result of separating aroma compounds by a supercritical fluid chromatogram equipped with a column containing silica gel chemically modified with a phenyl group as a filler. A chromatogram of the result of separating aroma compounds by a supercritical fluid chromatogram equipped with a column containing silica gel chemically modified with an aminopropyl group as a filler. A chromatogram as a result of separating an aroma compound by carrying out one embodiment of the method for separating an aroma compound according to the present invention by the supercritical fluid chromatograph of this example. A table summarizing the retention coefficients of each aroma compound obtained from the chromatograms obtained from this example and the conventional method for separating aroma compounds.

An example of a method for separating an aroma compound and an example of a supercritical fluid chromatograph (SFC) according to the present invention will be described below with reference to the drawings.

FIG. 1 is a configuration diagram of a main part of the supercritical fluid chromatograph of this embodiment. The supercritical fluid chromatograph 1 includes a cylinder 11 containing a mobile phase substance, a pressurizing 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 unit 16, and a column. 17, back pressure control valve 18, spectroscopic detector 19, and fraction collector 20 are provided. It also includes a control / processing unit 4 that controls the operation of each of these units.

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

The spectroscopic detector 19 transmits the light transmitted through the flow cell 191 into which the sample liquid eluted from the column 17 is introduced, the lamp 192 that irradiates the sample liquid flowing in the flow cell 191 with light having a wavelength in a predetermined range, and the flow cell 191. It is provided with a light detection unit 193 that separates wavelengths and measures the intensity of light of each wavelength.

The fraction collector 20 separates the aroma compound based on the detection result by the spectroscopic detector 19, and separates the aroma compound into the sorting container 21 and the effluent from the spectroscopic detector 19 into the sorting container 21. A sorting unit 22 is provided. The effluent that has passed through the preparative unit 22 is discarded in the drain.

The control / processing unit 4 includes a storage unit 41 including an aroma compound database 411, a measurement control unit 421 and an aroma 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 aroma 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.

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

When the user instructs the start of measurement of the aroma compound, the measurement control unit 421 displays a screen on the display unit 6 for the user to select the measurement target from the aroma compounds stored in the aroma compound database 411. indicate. When the user determines the aroma compounds to be measured by an appropriate operation, the measurement control unit 421 reads out the absorption spectrum information and the measurement conditions 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 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 to a predetermined pressure. Feed at 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.

Further, the pump 14 supplies the methanol contained in the liquid container 13 at a predetermined flow rate. Carbon dioxide and methanol are mixed by the mixer 15. In the sample injection unit 16, the sample is injected into the flow of the mobile phase formed by mixing carbon dioxide and methanol. The sample in this example contains a plurality of types of aroma compounds.

The sample injected into the mobile phase is introduced into the column 17 in the flow of the mobile phase. In column 17, each compound in the sample interacts with the stationary phase in column 17. Then, each compound is separated in time according to the magnitude and amount of interaction with the stationary phase, and flows out from the column 17.

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

The aroma compound identification unit 422 creates an absorption spectrum each time the measurement data is stored in the storage unit 41, and collates it with the absorption spectrum of the aroma compound to be measured read from the aroma compound database 411. When the two match with a predetermined accuracy or higher, the measurement control unit 421 outputs a control signal for causing the fraction collector 20 to separate the aroma compound. In addition, the aroma compound identification unit 422 sequentially determines whether or not a significant difference appears in the absorption spectrum. If the absorption spectra stored in the aroma compound database 411 do not match, but a significant difference appears in the absorption spectra, it is possible that an unknown aroma compound has flowed out from column 17. Therefore, the measurement control unit 421 outputs a control signal for causing the fraction collector 20 to separate the effluent from the column 17 even when it is determined that a significant difference appears in the absorption spectrum.

When a control signal instructing fractionation is output from the measurement control unit 421, the fraction collector 20 disposes of the aroma compound (or an outflow that may contain an unknown aroma compound) in the fraction collector 22. They are sorted into the sorting container 21 at the timing of reaching. When the sorting of one aroma compound or effluent is completed, the sorting unit 22 moves to the position of the sorting container 21 adjacent to the downstream side and waits for the control signal from the measurement control unit 421. In this way, the desired aroma compound and the unknown aroma compound are sequentially sorted into the sorting container 21. The effluent that flows out of the flow cell 191 of the spectroscopic detector 19 and has passed through the sorting unit 22 is discarded in the drain. Here, the case where both the aroma compound to be measured and the unknown aroma compound (the effluent that may be) are separated has been described, but only one of them is separated according to the purpose of measurement. You may do so.

Here, the method for sorting the aroma compound of this example and the column 17 used in the supercritical fluid chromatograph will be described.

In silica gel-based columns widely used in supercritical fluid chromatograms, highly polar silanol groups are present on the surface of silica gel chemically modified with functional groups. When an aroma compound, which is a low molecular weight compound, is introduced together with a supercritical fluid having high diffusivity, it passes through a stationary phase and penetrates to the surface of silica gel, and an undesired interaction with a silanol group existing on the surface occurs. Occurs. It is presumed that these undesired interactions are the reason why the aroma compounds could not be separated by the silica gel-based column in the past.

Since the aroma compound generally has a chain or cyclic unsaturated hydrocarbon structure, it is effective to use a compound having an unsaturated hydrocarbon structure as the stationary phase. The present inventor has proposed in Patent Documents 3 and 4 a column packed with polymer beads having an unsaturated hydrocarbon structure (hereinafter, referred to as "polymer beads"), and this column separates aroma compounds. I thought that it was also effective. That is, when polymer beads are used as the filler, undesired interactions are less likely to occur between the aroma compound to be separated and the filler, and the sharpness is higher than when silica gel or chemically modified silica is used. I thought that a good peak could be obtained.

Examples of the material of the polymer beads used for the filler of the column 17 include an acrylic polymer, a styrene polymer, a polyacrylamide polymer, a cellulosic polymer and the like. Supercritical fluid In a supercritical fluid chromatograph using carbon dioxide as a mobile phase, a pressure exceeding the critical pressure of carbon dioxide (7.4 MPa) is applied to the column 17 arranged in the analysis flow path, so that polymer beads as a filler Is required to have pressure resistance. Further, the polymer beads are required to have high solvent resistance so that swelling and shrinkage do not occur even when the mixing ratio of carbon dioxide and the modifier is changed. Therefore, as the polymer beads, those having a small degree of swelling with respect to a solvent are preferably used.

The polymer beads used as the column packing material preferably contain a crosslinked polymer. The polymer beads preferably have a swelling degree when absorbing tetrahydrofuran and a swelling degree when absorbing methanol are both 1.4 or less. By using such a polymer bead with a low swelling degree, a good peak shape can be obtained, and since the polymer bead has high durability, a good analysis result tends to be obtained even when repeated analysis is performed. Further, since the polymer beads having a low swelling degree can sufficiently increase the filling pressure at the time of filling the column, it is possible to suppress the deterioration of the analysis performance by the supercritical fluid chromatograph. Tetrahydrofuran is a substance that easily swells the resin material among the substances used as the mobile phase. Methanol is a substance widely used as a modifier. Therefore, by using polymer beads having a low degree of swelling for both of them as a filler, it is possible to perform measurement using various mobile phases (including those to which a modifier is added).

The degree of swelling of the polymer beads is determined based on the volume change before and after the polymer beads are dispersed in the solvent. The degree of swelling of the polymer beads when absorbing tetrahydrofuran and the degree of swelling when absorbing methanol are more preferably 1.3 or less, still 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 that a column having a high theoretical plate number can be easily obtained. 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, the value of the coefficient of variation (CV), which is an index indicating the dispersibility of the particle size (diameter) of the polymer beads, is preferably small, for example, 25% or less. It may be 20% or less, 15% or less, or 10% or less. The lower limit of the CV value is not particularly limited, but is generally 1% or more. The polymer beads may be classified using an arbitrary sieve or the like for the purpose of adjusting the average particle size and the CV value.

The higher the degree of cross-linking of the polymer, the smaller the degree of swelling of the polymer beads tends to be. 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. When the degree of cross-linking is within the above range, it is not easily affected by the supercritical fluid and the analysis performance can be improved. The degree of cross-linking of the cross-linked polymer is a blending ratio of the cross-linked monomer in the monomer used for polymerization, and is defined as a mass ratio of the cross-linked monomer based on the total mass of the polymerizable monomer.

The crosslinked monomer is a compound having two or more polymerizable functional groups, and is a divinyl compound such as divinylbenzene, divinylbiphenyl, or divinylnaphthalene; diallyl phthalate and its isomers; triallyl isocyanurate and its derivatives; polyfunctionality (meth). ) Acrylic acid ester and the like can be mentioned. The cross-linking monomer may be used alone or in combination of two or more. Examples of the polyfunctional (meth) acrylic acid ester include di (meth) acrylic acid ester and trifunctional or higher functional (meth) acrylic acid ester.

Examples of the di (meth) acrylic acid ester include alkanediol di (meth) acrylate in which two (meth) acrylates are bonded to an alkylene group. The alkylene group may have, for example, 1 to 20 or 1 to 5 carbon atoms. 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, and 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, Examples thereof include 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 include ethoxylated bisphenol A-based di (meth) acrylates, propoxylated ethoxylated bisphenol A-based di (meth) acrylates, tricyclodecanedimethanol di (meth) acrylates, 1, Di (meth) acrylates such as 1,1-trishydroxymethylethanedi (meth) acrylate, ethoxylated cyclohexanedimethanol di (meth) acrylate; and (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di Includes (poly) alkylene glycol di (meth) acrylates such as (meth) acrylates and (poly) tetramethylene glycol di (meth) acrylates.

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 and 1. Examples thereof include 1-trishydroxymethylethanetri (meth) acrylate and 1,1,1-trishydroxymethylpropanetriacrylate.

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

A monofunctional monomer may be used together with the crosslinked monomer. Examples of the monofunctional monomer 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, Methyl α-chloroacrylic acid, Methyl methacrylate, Ethyl methacrylate, propyl methacrylate, n-butyl methacrylate, Isobutyl methacrylate, methacrylate Monofunctional (meth) acrylic acid esters such as hexyl, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, 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-octyl styrene, pn-nonyl styrene, pn-decyl styrene, pn-dodecyl styrene, p-methoxy styrene, p-phenyl styrene, p-chloro styrene, 3,4-dichloro Styrene such as styrene and its derivatives; vinyl esters such as vinyl acetate, vinyl propionate, vinyl benzoate, vinyl butyrate; N-vinyl such as N-vinylpyrrole, N-vinylcarbazole, N-vinylindol, N-vinylpyrrolidone Compounds: Fluorinated monomers such as vinyl fluoride, vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, trifluoroethyl acrylate, 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 made of a crosslinked polymer, or may have a partially crosslinked polymer. From the viewpoint of reducing the degree of swelling, it is preferable that at least the outer layer of the polymer beads contains a crosslinked polymer. Polymer beads having a crosslinked polymer in the outer layer can be 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 tends to be. In general, polymer beads may have difficulty in forming particles having a smaller particle size than silica gel or the like, but the seed polymerization method tends to form particles having a smaller particle size.

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

The (meth) acrylate-based particles are obtained by polymerizing the (meth) acrylic acid ester. Examples of the (meth) acrylic acid ester include the (meth) acrylic acid ester having a linear or branched alkyl group exemplified above. Styrene-based particles can be obtained, for example, by polymerizing styrene-based monomers such as styrene, p-methylstyrene, p-chlorostyrene, chloromethylstyrene, and α-methylstyrene. As the monomer for obtaining seed particles, in addition to the above (meth) acrylic acid ester and styrene-based 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 an emulsion polymerization method, a soap-free emulsion polymerization method, or a dispersion polymerization method using the above-mentioned monomer. The average particle size of the seed particles may be adjusted according to the design particle size of the obtained 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 close to a true sphere. From these viewpoints, the average particle size of the seed particles is preferably 0.1 to 2.0 μm, more preferably 0.5 to 2.0 μm, and 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 obtained polymer beads. The CV value of seed particles is generally 1% or higher.

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 in 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 emulsion containing a polymerizable monomer and an aqueous medium to allow 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 it in the aqueous medium with a fine emulsifier such as a homogenizer, an ultrasonic processor, or a nanomizer. Examples of the aqueous medium include water or a mixed medium of water and a water-soluble solvent (for example, a lower alcohol). The aqueous medium may contain a surfactant. As the surfactant, any of anionic, cationic, nonionic and zwitterionic surfactants may be used.

The emulsion may contain a polymerization initiator such as an organic peroxide or an azo compound, 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 polymer dispersion stabilizer such as polyvinyl alcohol or polyvinylpyrrolidone in order to improve the dispersion stability of the seed particles. The polymer 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 a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble B vitamins, citric acid, and polyphenols. By including the polymerization inhibitor, the 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 where the seed particles are dispersed in an aqueous dispersion. For example, by stirring the emulsion after adding the seed particles at room temperature for 1 to 24 hours, the seed particles can absorb the polymerizable monomer. By heating the emulsion to about 30 to 50 ° C., the absorption of the polymerizable monomer tends to be promoted.

The mixing ratio of the polymerizable monomer to the seed particles is not particularly limited, but for example, from the viewpoint of efficiently producing polymer beads having a desired average particle size, 800 parts by mass or more or 1500 parts by mass with respect to 100 parts by mass of seed particles. It may be parts by mass or more. On the other hand, for example, from the viewpoint of suppressing the independent suspension polymerization of the polymerizable monomer in an aqueous medium and efficiently producing polymer beads having the desired average particle size, the mixing ratio of the polymerizable monomer is set. It may be 100,000 parts by mass or less or 35,000 parts by mass or less with respect to 100 parts by mass of seed particles. Since the seed particles swell by absorbing the polymerizable monomer, whether or not the absorption of the polymerizable monomer into the seed particles is completed by confirming the expansion of the particle size of the seed particles using an optical microscope can be determined. Can be judged.

Polymer beads are obtained by polymerizing the polymerizable monomer absorbed in the seed particles. The polymerization conditions are not particularly limited, and may be appropriately selected depending on the type of monomer and the like. After completion of the polymerization, the aqueous medium is removed from the polymerization solution by centrifugation or filtration, if necessary, washed with water and a solvent, and then dried to isolate the polymer beads.

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

In the present invention, the sample can be separated and analyzed in the same manner as the conventional supercritical fluid chromatograph except that the predetermined polymer beads are used as the filler of the separation column, and the structure of the supercritical fluid chromatograph is shown in the figure. It is not limited to the one shown in 1.

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

Next, a conventional supercritical fluid chromatograph (with a silica gel-based column) and a supercritical fluid chromatograph of this example (with a column loaded with polymer beads of styrene and divinylbenzene as a filler). The results of separating 23 types of aroma compounds using each of) will be described.

FIG. 2 is a chromatogram of the result of separating aroma compounds by a conventional supercritical fluid chromatograph equipped with a column containing silica gel chemically modified with an octadecyl group, and FIG. 3 is silica gel chemically modified with a phenyl group. Chromatogram of the result of separating aroma compounds by a conventional supercritical fluid chromatograph equipped with a column using the above, FIG. 4 shows a conventional chromatogram equipped with a column containing silica gel chemically modified with an aminopropyl group. It is a chromatogram of the result of separating aroma compounds by the supercritical fluid chromatograph of. On the other hand, FIG. 5 is a chromatogram of the result of separating the aroma compound by performing it by the supercritical fluid chromatograph of this example. FIG. 6 is a table summarizing the retention coefficients k'of each aroma compound obtained from the chromatograms of FIGS. 2 to 5.

In FIG. 2 (using silica gel chemically modified with an octadecyl group as a filler), all 23 types of aroma compounds flowed out during the retention time of 1.0 to 2.0 minutes, and were hardly separated. In FIG. 3 (using silica gel chemically modified with a phenyl group as a filler), the aroma compounds are separated as compared with FIG. 2, but all the aroma compounds are still in the retention time of 1.0 to 4.0 minutes. Is leaking out, and the separation is still insufficient. In FIG. 4 (using silica gel chemically modified with an aminopropyl group as a filler), aroma compounds are separated as compared with FIGS. 2 and 3, but a large number of aromas are separated during the retention time of 1.0 to 2.5 minutes. The sex compounds are solidified and separation is insufficient to separate the aroma compounds.

On the other hand, in the chromatogram of FIG. 5 obtained by the supercritical fluid chromatograph of this example, as can be clearly seen from the comparison with FIGS. 2 to 4, it can be seen that each aroma compound is separated. Although some peaks of the aroma compound may overlap for some time, the number is small and can be separated by changing the modifier or performing another chromatography after fractionation.

The above embodiment is an example, and can be appropriately modified according to the gist of the present invention.
In the above embodiment, in order to separate the aroma compound, a spectroscopic detector for detecting the aroma compound in a non-destructive manner is provided. However, when it is not necessary to separate the aroma compound, the aroma compound may not be separated. A mass spectrometer may be provided in place of the spectroscopic detector (or in addition to the spectroscopic detector). Further, with respect to the aroma compounds in which some of the peaks overlap in the chromatograph shown in FIG. 5, they may be collected by the fraction collector 20 and then separated by another chromatograph.

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

(First aspect)
In the method for separating an aroma compound according to the first aspect of the present invention, a sample containing an aroma compound is placed on a flow of a mobile phase, which is a supercritical fluid of a predetermined substance, from a polymer having an unsaturated hydrocarbon structure. Including the step of introducing a column loaded with the filler.

In the method for separating aroma compounds according to the first aspect of the present invention, the aroma compounds are separated using a column loaded 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 a functional group in a conventionally used column. The reason why the aroma compounds could not be separated by the silica gel-based column was that the aroma compounds, which are low molecular weight compounds introduced into the column together with the highly diffusible supercritical fluid, passed through the stationary phase of the silica gel. It is considered that this is because it penetrated to the surface and an undesired interaction with the silanol group existing on the surface occurred. On the other hand, in the present invention, a column loaded with a filler made of a polymer of a compound having an unsaturated hydrocarbon structure, which is a stationary phase, is used. That is, the entire filler functions as a stationary phase and does not contain functional groups that cause undesired interactions, so that the aroma compound can be separated.

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

In the method for separating an aroma compound according to the second aspect of the present invention, carbon dioxide in a supercritical state mainly constitutes a mobile phase. 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 not flammable or chemically reactive, so it can be measured safely. In addition, high-purity products can be obtained at low cost.

(Third aspect)
The method for separating an aroma compound according to a third aspect of the present invention is the method for separating an aroma compound according to the first or second aspect.
The filler is polymer beads
The polymer beads have a swelling degree when absorbing tetrahydrofuran and a swelling degree when absorbing methanol are both 1.4 or less.
The polymer beads contain a crosslinked polymer.

Tetrahydrofuran is a substance that easily swells a resin material among substances used as a mobile phase. Methanol is a substance widely used as a modifier. In the method for separating aroma compounds according to the third aspect of the present invention, since a column loaded with polymer beads having a low swelling degree to both tetrahydrofuran and methanol as a filler is used, various mobile phases (modifiers added) are used. Including) can be used for measurement. Also, the bonds within the crosslinked polymer are stronger than those of 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.

(Fourth aspect)
The method for separating an aroma compound according to a fourth aspect of the present invention is the method for separating an aroma 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 aroma compound according to the fourth aspect of the present invention, a crosslinked polymer having at least one selected from the group consisting of structural units derived from divinylbenzene and structural units derived from di (meth) acrylic acid ester is used. By using the polymer, a polymer having a high crosslink density (a polymer having a high ratio of a polyfunctional monomer-derived structure) can be easily obtained, and the degree of swelling of the polymer beads can be reduced.

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

As described above, the degree of swelling is reduced by using a polymer having crosslinks, and the higher the degree of cross-linking of the polymer, the smaller the degree of swelling of the polymer beads tends to be. In the method for separating an aroma compound 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 crosslink of 50% or more.

(6th aspect)
The method for separating an aroma compound according to a sixth aspect of the present invention is the method for separating an aroma compound according to any one of the first to fifth aspects.
The average particle size of the polymer beads is 1 to 10 μm.

In the method for separating an aroma compound 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 chance of interaction between the aroma compound and the stationary phase is increased, and the number of theoretical plates of the column is increased. Can be enhanced. Further, by setting the average particle size of the polymer beads to 1 μm or more, it is possible to suppress an excessive increase in pressure in the column during use.

(7th aspect)
In the method for separating aroma compounds according to the seventh aspect of the present invention, the aroma compounds separated in the methods for separating aroma compounds according to the first to sixth aspects are further analyzed by chromatography and / or mass spectrometry. To do.

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

(8th aspect)
The supercritical fluid chromatograph according to the eighth aspect of the present invention is
An aroma compound database containing measurement data of a plurality of aroma compounds acquired by a predetermined measurement method, and
A column loaded with a filler made of a polymer having an unsaturated hydrocarbon structure,
A supercritical fluid feeding unit that feeds a supercritical fluid of a predetermined substance to the column,
A sample injection unit provided in the flow path from the supercritical fluid feeding unit to the column and injecting a sample into the supercritical fluid,
A measuring unit that sequentially measures the effluent from the column by the predetermined measuring method,
It is provided with an aroma compound identification unit that identifies the aroma compound contained in the effluent by collating the measurement data obtained by the measurement with the measurement data recorded in the database of the aroma compound.

By using the supercritical fluid chromatograph according to the eighth aspect of the present invention, the aroma compound can be sufficiently separated as in the first aspect. In addition, by identifying the compound after separation, if the sample contains an unknown aroma compound, the possibility can be easily grasped.

1 ... Supercritical fluid chromatograph 11 ... Cylinder 12 ... Pressurized pump 13 ... Liquid container 14 ... Pump 15 ... Mixer 16 ... Sample injection unit 17 ... Column 171 ... Column oven 18 ... Back pressure control valve 19 ... Spectral detector 191 ... Flow cell 192 ... Lamp 193 ... Light detection unit 20 ... Fraction collector 21 ... Sorting container 22 ... Sorting unit 4 ... Control / processing unit 41 ... Storage unit 411 ... Aroma compound database 421 ... Measurement control unit 422 ... Aroma compound identification Unit 5 ... Input unit 6 ... Display unit

Claims (8)

Aroma, including the step of introducing a sample containing an aroma compound into a column loaded with a filler made of a polymer having an unsaturated hydrocarbon structure by placing it in a mobile phase flow which is a supercritical fluid of a predetermined substance. Method for separating sex compounds. The method for separating an aroma compound according to claim 1, wherein the mobile phase is carbon dioxide in a supercritical state or carbon dioxide in a supercritical state with a modifier added. The filler is polymer beads
The polymer beads have a swelling degree when absorbing tetrahydrofuran and a swelling degree when absorbing methanol are both 1.4 or less.
The method for separating an aroma compound according to claim 1 or 2, wherein the polymer beads contain a crosslinked polymer. The method for separating an aroma compound according to claim 3, wherein the crosslinked polymer has at least one selected from the group consisting of a structural unit derived from divinylbenzene and a structural unit derived from di (meth) acrylic acid ester. The method for separating an aroma compound according to claim 3 or 4, wherein the degree of cross-linking of the cross-linked polymer is 50% or more. The method for separating an aroma compound according to any one of claims 1 to 4, wherein the polymer beads have an average particle size of 1 to 10 μm. A method for analyzing an aroma compound, which comprises analyzing the aroma compound separated for each component by the method according to any one of claims 1 to 6 by chromatography and / or mass spectrometry. An aroma compound database containing measurement data of a plurality of aroma compounds acquired by a predetermined measurement method, and
A column loaded with a filler made of a polymer having an unsaturated hydrocarbon structure,
A supercritical fluid feeding unit that feeds a supercritical fluid of a predetermined substance to the column,
A sample injection unit provided in the flow path from the supercritical fluid feeding unit to the column and injecting a sample into the supercritical fluid,
A measuring unit that sequentially measures the effluent from the column by the predetermined measuring method,
Supercritical including an aroma compound identification unit that identifies an aroma compound contained in the effluent by collating the measurement data obtained by the measurement with the measurement data recorded in the aroma compound database. Fluid chromatograph.

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