CN112098528B

CN112098528B - Method for separating aroma compound and supercritical fluid chromatograph

Info

Publication number: CN112098528B
Application number: CN202010072189.0A
Authority: CN
Inventors: 藤户由佳; 马场健史
Original assignee: Shimadzu Corp
Current assignee: Shimadzu Corp
Priority date: 2019-06-17
Filing date: 2020-01-21
Publication date: 2023-08-08
Anticipated expiration: 2040-01-21
Also published as: JP2020204514A; CN112098528A; JP7306090B2

Abstract

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

Description

Method for separating aroma compound and supercritical fluid chromatograph

Technical Field

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

Background

Conventionally, in order to analyze an unknown aroma compound contained in a natural substance, a gas chromatograph is used (for example, patent document 1). In the analysis of aroma compounds by a gas chromatograph, a sample extracted from a natural substance is vaporized and then introduced into a column by a gas flow carried in a carrier gas, and separated from other compounds. The isolated compound can be analyzed for structure, for example, by measurement using a nuclear magnetic resonance (NMR: nuclear Magnetic Resonance) apparatus.

However, since the capillary column used in the gas chromatograph generally coats the stationary phase inside the capillary tube, the amount of the compound that can be adsorbed to the stationary phase inside the capillary column is small. In NMR, at least several μg of a sample is required for one measurement, but the amount of a compound that can be separated at one time is several ng in a gas chromatograph, so in the case of using a gas chromatograph, the amount of a compound required for structural analysis by NMR cannot be obtained without repeating the separation a plurality of times.

In recent years, analysis using a supercritical fluid chromatograph has been attempted. In a supercritical fluid chromatograph, a supercritical fluid such as carbon dioxide is used as a mobile phase, and the mobile phase injected with a sample is introduced into a packed column packed with a filler containing a stationary phase to separate a compound. In the supercritical fluid chromatograph, since a supercritical fluid having low viscosity and high diffusivity is used as a mobile phase, a large amount of compounds can be separated at one time by a packed column while maintaining the same advantages as those of the gas chromatograph such as high flow rate and high resolution.

In the supercritical fluid chromatograph, since the supercritical fluid is used as a mobile phase, the internal flow path must be maintained at a high pressure. Therefore, the pressure resistance of the column is required to be low with respect to the swelling property of the supercritical fluid. Accordingly, silica gel-based columns satisfying these factors are widely used in supercritical fluid chromatographs. The column of the silica gel matrix is packed with a functional group of a type corresponding to the property of the target compound as a filler, the functional group being chemically modified on the surface of the silica gel. For example, patent document 2 describes a column having octadecyl or the like as a matrix of silica gel (ODS: ostade-cylilane) to which silica gel is chemically modified as a solid phase.

Prior art literature

Patent literature

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

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

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

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

Disclosure of Invention

Technical problem to be solved by the invention

Patent document 2 shows an example of separating vitamins using a supercritical fluid chromatograph having a chromatographic column with a silica gel matrix. However, it was found that when separation of the aroma compounds was attempted using a supercritical fluid chromatograph employing a chromatographic column with a silica gel matrix, the separation could not be performed sufficiently.

The technical problem to be solved by the invention is to provide a technology for separating aroma compounds by using a supercritical fluid chromatograph.

Solution to the above technical problems

The separation method of aroma compounds of the present invention, which has been completed to solve the above-described problems, comprises the following steps: a sample containing a fragrant compound is placed on a fluid stream of a mobile phase, which is a supercritical fluid of a predetermined substance, and introduced into a column packed with a packing material composed of a polymer having an unsaturated hydrocarbon structure.

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

a fragrance compound database storing a plurality of measurement data of fragrance compounds obtained by a predetermined measurement method;

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

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

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

a measuring unit configured to sequentially measure an effluent from the chromatographic column by using the predetermined measurement method;

and a fragrance compound identification unit configured to compare measurement data obtained by the measurement with measurement data recorded in the database of fragrance compounds, and identify fragrance compounds contained in the effluent.

Effects of the invention

The aroma compound may be defined as a compound having a molecular weight of 800 or less, for example, having a chain or cyclic unsaturated hydrocarbon structure. The aroma compound has a chain or cyclic unsaturated hydrocarbon structure, so that it has a low polarity, and most of the aroma compound is a low molecular compound having a molecular weight of 800 or less, so that it has weak intermolecular bonds and volatility. Thus, it can also be defined as a volatile low molecular compound.

The unsaturated hydrocarbon structure may be either chain or cyclic, or both of them may be contained.

In the method for separating aroma compounds and the supercritical fluid chromatograph of the present invention, aroma compounds are separated using a chromatographic column packed with a filler composed of a polymer having an unsaturated hydrocarbon structure. The reason why the aroma compound cannot be separated by the column of the silica gel matrix is that the aroma compound, which is a low molecular compound introduced into the column together with the supercritical fluid having high diffusivity, passes through the stationary phase to enter the surface of the silica gel, and an undesired interaction occurs between the aroma compound and the silanol groups having high polarity present on the surface. In contrast, in the present invention, a column packed with a packing composed of a polymer of a compound having an unsaturated hydrocarbon structure as a stationary phase is used. That is, since the filler as a whole acts as a stationary phase and does not contain a functional group that causes undesired interaction, the aroma compound can be sufficiently separated.

Drawings

FIG. 1 is a schematic diagram of the supercritical fluid chromatograph according to an embodiment of the present invention.

Fig. 2 is a chromatogram of the result of separation of aroma compounds by a supercritical fluid chromatograph equipped with a chromatographic column using octadecyl chemically modified silica gel as a filler.

Fig. 3 is a chromatogram of the result of separation of aroma compounds by a supercritical fluid chromatograph equipped with a chromatographic column using phenyl chemically modified silica gel as a filler.

Fig. 4 is a chromatogram of the result of separation of aroma compounds by a supercritical fluid chromatograph equipped with a chromatographic column using silica gel chemically modified with aminopropyl as a filler.

Fig. 5 is a chromatogram showing the result of separation of aroma compounds according to one embodiment of the method for separating aroma compounds of the present invention by supercritical fluid chromatography according to this example.

Fig. 6 is a table summarizing retention coefficients of the respective aroma compounds obtained by the chromatogram obtained by the separation method of the aroma compounds of this example and the conventional method.

Detailed Description

An example of the method for separating aroma compounds and supercritical fluid chromatograph (SFC: supercritical Fluid Chromatography) according to the present invention will be described below with reference to the accompanying drawings.

Fig. 1 is a main part configuration diagram of a supercritical fluid chromatograph according to the present embodiment. The supercritical fluid chromatograph 1 includes: a gas cylinder 11 containing a mobile phase substance; a pressurizing pump 12; a liquid container 13 containing a modifier solution added to the mobile phase; a pump 14; a mixer 15; a sample injection section 16; a chromatographic column 17; a back pressure control valve 18; a spectroscopic detector 19 and a fraction collector 20. The control processing unit 4 is provided to control the operations of these units.

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

The spectroscopic detector 19 includes: a flow cell 191 into which a sample liquid eluted from the column 17 is introduced; a lamp 192 for irradiating the sample liquid flowing through the flow cell 191 with light having a wavelength within a predetermined range; and a light detection unit 193 that performs wavelength separation on the light transmitted through the flow cell 191 and measures the intensity of the light at each wavelength.

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

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

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

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

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

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

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

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

Each time the measurement data is stored in the storage unit 41, the aroma compound identification unit 422 generates an absorption spectrum, and compares the absorption spectrum with the absorption spectrum of the aroma compound of the measurement target read from the aroma compound database 411. If the two are identical to each other at a level equal to or higher than a predetermined level of accuracy, the measurement control unit 421 outputs a control signal for fractionating the aroma compound into the fraction collector 20. The aroma compound identifying unit 422 also sequentially determines whether or not an intentional difference has occurred in the absorption spectrum. If the absorption spectra stored in the aroma compound database 411 are inconsistent, but an intentional difference occurs in the absorption spectra, an unknown aroma compound may flow out from the chromatographic column 17. Then, even when it is determined that an intentional difference has occurred in the absorption spectrum, the measurement control section 421 outputs a control signal for fractionating the effluent from the chromatographic column 17 to the fraction collector 20.

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

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

In a column of a silica gel matrix widely used in a supercritical fluid chromatograph, highly polar silanol groups exist on the surface of silica gel chemically modified with functional groups. When the aroma compound, which is a low molecular compound, is introduced together with a supercritical fluid having high diffusivity, the aroma compound enters the surface of the silica gel through the stationary phase, and undesirable interactions with silanol groups present on the surface occur. It is assumed that the failure to separate aroma compounds by chromatography on a silica gel matrix is due to such undesired interactions.

In general, since the aroma compound has a chain or cyclic unsaturated hydrocarbon structure, it is effective to use a compound having an unsaturated hydrocarbon structure as a stationary phase. In consideration of the inventors, patent document 3 and patent document 4 propose a column packed with beads of a polymer having an unsaturated hydrocarbon structure (hereinafter referred to as "polymer beads"), which is also effective for separation of aroma compounds. That is, it is considered that when polymer beads are used as the filler, an unexpected interaction between the aroma compound to be separated and the filler is less likely to occur than when silica gel or chemically modified silica is used, and a sharp peak can be obtained.

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

The polymer beads used as column packing preferably contain crosslinked polymers. The polymer beads preferably have a swelling degree of 1.4 or less when they absorb tetrahydrofuran and a swelling degree when they absorb methanol. By using such polymer beads having a low degree of swelling, a good peak shape can be obtained, and the durability of the polymer beads is high, so that even in the case of performing repeated analysis, there is a tendency that a good analysis result can be obtained. In addition, since the polymer beads having a low swelling degree can sufficiently increase the packing pressure at the time of packing into the chromatographic column, the decrease in analytical performance caused by the supercritical fluid chromatograph can be suppressed. Among the substances used as the mobile phase, tetrahydrofuran is a substance that swells the resin material particularly easily. Further, methanol is a substance widely used as a modifier. Therefore, by using polymer beads having low swelling relative to both as a filler, various mobile phases (including a mobile phase to which a modifier is added) can be used for measurement.

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

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

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

The higher the degree of crosslinking of the polymer, the less the swelling of the polymer beads tends to be. The cross-linking degree 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 crosslinking degree is within the above range, the influence of the supercritical fluid is less likely to occur, and the analytical performance can be improved. The crosslinking degree of the crosslinked polymer is the blending ratio of the crosslinking monomer among the monomers used for polymerization, and is defined as the mass ratio of the crosslinking monomer based on the total mass of the polymerizable monomers.

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

As the di (meth) acrylate, there may be mentioned two alkylene glycol di (meth) acrylates in which the (meth) acrylate is bonded to an alkylene group. The carbon number of the alkyl group may be, for example, 1 to 20 or 1 to 5. The alkylene group may be any of linear, branched or cyclic. The alkylene group may have a substituent such as a hydroxyl group.

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

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

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

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

Crosslinking monomers and monofunctional monomers may also be used simultaneously. Examples of the monofunctional monomer include monofunctional (meth) acrylates such as methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, isobutyl acrylate, hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, dodecyl acrylate, lauryl acrylate, stearyl acrylate, 2-chloroethyl acrylate, phenyl acrylate, α -methyl chloroacrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, and stearyl methacrylate; styrene such as styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α -methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2, 4-dimethylstyrene, p-n-butylstyrene, p-t-butylstyrene, p-n-hexylstyrene, p-n-octylstyrene, p-n-nonylstyrene, p-n-decylstyrene, p-n-dodecylstyrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, 3, 4-dichlorostyrene and the like, and derivatives thereof; vinyl esters such as vinyl acetate, vinyl propionate, vinyl benzoate, and vinyl butyrate; n-vinyl compounds such as N-vinyl pyrrole, N-vinyl carbazole, N-vinyl indole, and N-vinyl pyrrolidone; fluoromonomers such as ethylene fluoride, vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, trifluoroethyl acrylate, and tetrafluoropropyl acrylate; conjugated dienes such as butadiene and isoprene. These may be used singly or in combination of two or more.

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

Generally, the smaller the particle diameter of the beads, the larger the theoretical number of stages of the column. In general, although polymer beads are sometimes difficult to form particles having a smaller particle diameter than silica gel or the like, seed polymerization methods tend to form particles having a smaller particle diameter.

The seed polymerization method is a method in which seed particles are swelled in an emulsion containing a polymerizable monomer, and the polymerizable monomer is polymerized after the seed particles absorb the polymerizable monomer. Examples of the seed particles include (meth) acrylic acid ester particles and styrene particles.

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

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

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

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

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

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

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

The emulsion may contain water-soluble polymerization inhibitors such as nitrite, sulfite, hydroquinone, ascorbic acid, water-soluble vitamin B, citric acid, and polyphenols. By including a polymerization inhibitor, emulsion polymerization of monomers in an 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 to which the seed particles are added at room temperature for 1 to 24 hours. The emulsion tends to promote absorption of the polymerizable monomer by heating to about 30 to 50 ℃.

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

The polymer beads can be obtained by polymerizing the polymerizable monomer absorbed by the seed particles. The polymerization conditions are not particularly limited as long as they are appropriately selected according to the kind of monomer and the like. After the polymerization, the aqueous medium is removed from the polymerization solution by centrifugation or filtration as needed, and washed with water and a solvent and then dried, thereby separating polymer beads.

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

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

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 at the time of packing the polymer beads into the column may be, for example, 10MPa or more or 15MPa or more. By increasing the packing pressure, tailing of peaks in the chromatogram of the supercritical fluid chromatograph is suppressed, and a good peak shape is easily obtained. From the viewpoint of suppressing the change of the polymer beads or the breakage of the column, the packing pressure may be, for example, 60MPa or less or 50MPa or less.

Next, the results of separating 23 aroma compounds using a conventional supercritical fluid chromatograph (a chromatographic column having a silica gel matrix) and a supercritical fluid chromatograph of this example (a chromatographic column having polymer beads packed with styrene and divinylbenzene as a filler) will be described.

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

In fig. 2 (silica gel chemically modified with octadecyl is used as a filler), all of the 23 aroma compounds flow out during the holding time of 1.0 to 2.0 minutes, and are hardly separated. In fig. 3 (silica gel chemically modified with phenyl is used as a filler), the aroma compounds are separated, but all the aroma compounds still flow out in a period of 1.0 to 4.0 minutes, and the separation is still insufficient, as compared with fig. 2. In fig. 4 (silica gel chemically modified with aminopropyl is used as a filler), the aroma compounds are separated, but most aroma compounds solidify between 1.0 and 2.5 minutes in the holding time, and the separation is insufficient to fractionate the aroma compounds, as compared with fig. 2 and 3.

On the other hand, in the chromatogram of fig. 5 obtained by the supercritical fluid chromatograph of the present example, it is apparent from comparison with fig. 2 to 4 that each aroma compound is separated. Although the amount of time for which a part of peaks of the aroma compound overlap is small, the aroma compound can be separated by changing the modifier or performing other chromatography after fractionation.

The above-described embodiments are examples, and can be modified as appropriate according to the gist of the present invention.

In the above-described embodiment, the fractionating device for detecting the aroma compound in a nondestructive manner is provided for fractionating the aroma compound, but in the case where the fractionating of the aroma compound is not required, a mass analyzer may be provided instead of (or in addition to) the fractionating device. In the chromatogram shown in fig. 5, the aroma compounds having partially overlapped peaks may be extracted by the fraction collector 20 and then separated by another chromatograph.

Scheme (scheme)

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

(scheme 1)

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

In the method for separating a fragrant compound according to the 1 st aspect of the present invention, a chromatographic column packed with a packing material composed of a polymer having an unsaturated hydrocarbon structure is used to separate a fragrant compound. In chromatographic columns used in the past, highly polar silanol groups are present on the surface of silica gel chemically modified with functional groups. The reason why the aroma compound cannot be separated by the column of the silica gel matrix is that the aroma compound, which is a low molecular compound introduced into the column together with the supercritical fluid having high diffusivity, passes through the stationary phase to enter the surface of the silica gel, and an undesired interaction occurs between the aroma compound and silanol groups present on the surface. In contrast, in the present invention, a column packed with a packing composed of a polymer of a compound having an unsaturated hydrocarbon structure as a stationary phase is used. That is, since the filler as a whole functions as a stationary phase and does not contain a functional group that can cause undesired interaction, the aroma compound can be separated.

(scheme 2)

In the method for separating a fragrant compound according to claim 2 of the present invention, in the method for separating a fragrant compound according to claim 1,

the mobile phase is supercritical carbon dioxide or a substance with a modifier added to the supercritical carbon dioxide.

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

(scheme 3)

The method for separating a fragrant compound according to claim 3 of the present invention, in the method for separating a fragrant compound according to claim 1 or 2,

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

the polymer beads have a swelling degree of 1.4 or less when they absorb tetrahydrofuran and a swelling degree when they absorb methanol,

the polymer beads comprise crosslinked polymer.

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

(scheme 4)

In the method for separating a fragrant compound according to claim 4 of the present invention, in the method for separating a fragrant compound according to claim 3,

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

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

(scheme 5)

The method for separating a fragrant compound according to claim 5 of the present invention, in the method for separating a fragrant compound according to claim 3 or 4,

the cross-linking degree of the cross-linked polymer is more than 50%.

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

(scheme 6)

The method for separating a fragrant compound according to claim 6 of the present invention is the method for separating a fragrant compound according to any one of the above-mentioned 1 st to 5 th aspects,

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

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

(scheme 7)

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

If chromatographic analysis is performed in the method for separating a fragrant compound according to claim 7 of the present invention, even if the separation is not complete in the methods for separating a fragrant compound according to the above-mentioned 1 st to 6 th aspects, the separation can be made more complete. Further, by performing mass analysis, more detailed information such as the molecular structure of the aroma compound can be obtained.

(scheme 8)

The supercritical fluid chromatograph according to the 8 th aspect of the present invention includes:

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

Description of the reference numerals

1. Supercritical fluid chromatograph

11. Gas cylinder

12. Pressure pump

13. Liquid container

14. Pump with a pump body

15. Mixer

16. Sample injection part

17. Chromatographic column

171. Column temperature box

18. Back pressure control valve

19. Spectroscopic detector

191. Flow cell

192. Lamp with light-emitting device

193. Photodetector unit

20. Fraction collector

21. Fractionation vessel

22. Fractionation unit

4. Control processing unit

41. Storage unit

411. Aroma chemical database

421. Measurement control unit

422. Aroma chemical identification part

5. Input unit

6. And a display unit.

Claims

1. A method for separating a fragrant compound, comprising the steps of:

introducing a fluid stream of a mobile phase, which is a supercritical fluid containing a plurality of samples of aroma compounds on a predetermined substance, into a column packed with a filler composed of a polymer having an unsaturated hydrocarbon structure,

the aroma compound has a chain or cyclic unsaturated hydrocarbon structure and a molecular weight of 800 or less,

the polymer with unsaturated hydrocarbon structure in the filler is acrylic polymer, styrene polymer, polyacrylamide polymer and cellulose polymer.

2. A method for separating aroma compounds according to claim 1,

3. A method for separating aroma compounds according to claim 1,

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

the polymer beads comprise crosslinked polymer.

4. A method for separating aroma compounds according to claim 3,

5. A method for separating aroma compounds according to claim 3,

the cross-linking degree of the cross-linked polymer is more than 50%.

6. A method for separating aroma compounds according to claim 3,

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

7. A method for analyzing an aroma compound, characterized by comprising,

an aroma compound separated according to the component by the method of claim 1 is analyzed by chromatography and/or mass spectrometry.

8. A supercritical fluid chromatograph is characterized by comprising:

a fragrance compound database storing a plurality of measurement data of fragrance compounds obtained by a predetermined measurement method using a supercritical fluid chromatograph;