JP2010077086A - Method of separating capsaicinoids, method of separating capsinoids and method of separating capsaicinoids and capsinoids - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an industrially efficient and safe method of separating capsaicinoids, a method of separation capsinoids, and a method of separating capsinoids and capsaicinoids. <P>SOLUTION: The method of separating capsaicinoids from a raw material containing capsaicinoids by liquid chromatography having a stationary phase and a mobile phase comprises using porous organic polymer particles composed of a (meth)acrylic ester based crosslinked (co)polymer as the stationary phase. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for separating capsaicinoids and capsinoids by liquid chromatography. Specifically, a method for separating capsaicinoids or capsinoids from capsaicinoids or a raw material containing capsinoids by a liquid chromatography method, particularly a normal phase liquid chromatography method, and further a raw material containing capsaicinoids and capsinoids The present invention relates to a method for separating capsaicinoids and capsinoids from each other.

  Pepper is a plant that is widely used around the world as a food, spice and pharmaceutical ingredient. Its main pungent components are capsaicin and dihydrocapsaicin. Furthermore, there are nordihydrocapsaicin, homocapsaicin, homodihydrocapsaicin and the like as trace components, which are collectively referred to as capsaicinoids.

  In addition, for example, in some peppers such as “CH-19 sweet”, there are capsaicinoid-like substances collectively called capsinoids such as capsiate, dihydrocapsiate, nordihydrocapsiate, and capsaicinoids are hardly contained. Some do not exhibit a pungent taste (Non-Patent Documents 1 and 2). Capsinoids have effects such as energy metabolism promoting action, obesity suppressing action, and immunostimulatory action similar to those of capsaicinoids, and are expected to be used as functional foods, cosmetics, or pharmaceuticals like capsaicinoids.

  Various techniques have been disclosed so far for methods for separating these capsaicinoids or capsinoids from raw materials. Patent Document 1 is a method for extracting and separating capsinoid compounds from chili using an extraction / separation apparatus, and is one kind selected from the group consisting of chili pulverized product and water, acetone, alcohols, hexane, ether and dioxane, or Two or more kinds of extraction solvents are stirred and mixed in an inner mixer tank of the extraction / separation apparatus to extract a capsinoid compound in chili in the extraction solvent, and in the outer separation tank of the extraction / separation apparatus, Pulverizing the chili pulverized material from the mixture of the pepper pulverized material and capsinoid compound-containing extraction solvent sent from the outlet of the inner mixer tank, and discharging the pulverized chili powder from the discharge port of the outer separation tank; The technique regarding the extraction separation method of the capsinoid compound from the chili pepper characterized by including is disclosed.

  Patent Document 2 is a method for extracting and separating capsinoid compounds in chili in the mixed extraction solvent by bringing the ground pepper into contact with a mixed extraction solvent containing water and acetone. A technique relating to a method for extracting and separating capsinoid compounds from chili pepper, characterized in that a mixed extraction solvent in which a mixing molar ratio of water and acetone is set to a certain range is used as an extraction solvent.

  Patent Document 3 discloses a technique relating to a capsaicinoid-like substance having a specific chemical structure. Regarding the method for separating the capsaicinoid-like substance, in the Examples, oleoresin obtained by extracting capsinoid from chili pepper non-paste-fixed variety "CH-19 sweet" using ethyl acetate and distilling off ethyl acetate from the resulting extract A method of further purifying (resin oil) stepwise by silica gel chromatography and reverse phase chromatography on silica gel to obtain the desired capsaicinoid-like substance is disclosed.

  Patent Document 4 discloses a technique relating to a method for extracting a capsaicinoid-like substance, characterized in that the extraction process is performed using carbon dioxide in a supercritical state or in the vicinity thereof as an extractant.

  Patent Document 5 includes at least a solvent extraction step for extracting the capsinoid with a solvent from a capsioid-containing pepper, and a concentration step for separating the solvent and concentrating the capsinoid from the extract after the solvent extraction step. In the method for producing a capsinoid extract, a technique relating to a method for producing a capsinoid extract is disclosed, wherein ethanol or hydrous ethanol is used as the extraction solvent, and the solvent extraction step is performed under acidic conditions.

JP 2004-66227 A JP 2006-199604 A Japanese Patent Laid-Open No. 11-246478 Japanese Patent Laid-Open No. 2002-226445 JP 2004-18428 A J. et al. Agric. Food Chem. 46, 1695-1697 J. et al. Nat. Prod. 62, 335-336

  However, although Patent Documents 1 and 2 exemplify an adsorption method using an adsorbent such as a porous resin as a method for recovering the extract, strictly speaking, it is only extraction of capsinoids, and specific separation and purification. Since no example is disclosed, it is conceivable that the disclosed technique cannot always be applied as it is in a raw material mixture having a low level of separation and purification and capsaicinoids.

  In addition, the technique of stepwise purification by silica gel chromatography and reverse phase chromatography on silica gel disclosed in Patent Document 3, as pointed out in Patent Document 4, gradually decomposes capsinoids in the purification process. Therefore, there is a problem that capsinoids cannot be extracted with high yield.

  Furthermore, the extraction method disclosed in Patent Document 4 has a problem that the device cost of the supercritical extraction device used in the method is high.

  In addition, the technique disclosed in Patent Document 5 has a problem that an organic acid or an inorganic acid added to suppress decomposition of capsinoids is mixed in the extract.

Thus, in the prior art, it was difficult to separate capsaicinoids and capsinoids by an industrially efficient and stable method.
The present invention has been made in view of the above circumstances, and its object is to provide an industrially efficient and stable method for separating capsaicinoids, a method for separating capsinoids, and a method for separating capsaicinoids and capsinoids. There is to do.

  The method for separating capsaicinoids from a raw material containing capsaicinoids of the present invention is a method of separating by a liquid chromatography method having a stationary phase and a mobile phase. ) It is characterized by using porous organic polymer particles made of a polymer.

  In the separation method having such a configuration, capsaicinoids are obtained by using porous organic polymer particles made of a (meth) acrylic acid ester-based crosslinked (co) polymer that easily retains capsaicinoids as the stationary phase. Only desired capsaicinoids can be efficiently separated from the contained raw materials. Therefore, the separation method of the present invention can efficiently and stably perform high-efficiency separation of capsaicinoids, which has been difficult with the prior art, even on an industrial scale.

  The method for separating capsinoids from a raw material containing capsinoids according to the present invention is a method of separating by liquid chromatography having a stationary phase and a mobile phase. ) It is characterized by using porous organic polymer particles made of a polymer.

  In the separation method having such a configuration, capsinoids are obtained by using porous organic polymer particles made of a (meth) acrylic acid ester-based crosslinked (co) polymer that easily retains capsinoids as the stationary phase. Only the desired capsinoids can be efficiently separated from the contained raw material. Therefore, the separation method of the present invention can efficiently and stably perform high-efficiency separation of capsinoids, which has been difficult with the prior art, even on an industrial scale.

  The method for separating capsaicinoids and capsinoids from a raw material containing capsaicinoids and capsinoids according to the present invention is a method of separating by a liquid chromatography method having a stationary phase and a mobile phase. ) It is characterized by using porous organic polymer particles made of an acrylic ester-based crosslinked (co) polymer.

  The separation method having such a configuration uses, as the stationary phase, porous organic polymer particles made of a (meth) acrylic ester-based crosslinked (co) polymer that easily retains capsaicinoids and capsinoids, Only the desired capsaicinoids and capsinoids can be efficiently separated from the raw materials containing capsaicinoids and capsinoids, respectively. Therefore, the separation method of the present invention can efficiently and stably perform high-efficiency separation of capsaicinoids and capsinoids from the same raw material, which is difficult with the prior art, even on an industrial scale. .

  In the separation method of the present invention, the mobile phase preferably contains no water.

  In the separation method having such a configuration, since the mobile phase does not contain water, capsaicinoids and / or capsinoids are not hydrolyzed during the separation by liquid chromatography, and the desired capsaicinoid is efficiently produced. And / or capsinoids can be obtained.

  In the separation method of the present invention, the mobile phase has a lower relative dielectric constant than the porous organic polymer particles, or the solvent having the lower relative dielectric constant and the porous organic polymer particles. And any one of a mixed solvent with a solvent having a high relative dielectric constant.

  In such a separation method, the mobile phase has a solvent having a relative dielectric constant lower than that of the stationary phase, or a solvent having a relative dielectric constant lower than that of the stationary phase and a ratio higher than that of the stationary phase. By using one of the mixed solvents with the solvent having a dielectric constant, elution of capsaicinoids and / or capsinoids from the stationary phase can be controlled appropriately, and the separation performance of normal phase liquid chromatography can be improved. Can be improved.

  In the separation method of the present invention, the mobile phase is a solvent having a relative dielectric constant of less than 2.5, or a solvent having a relative dielectric constant of less than 2.5 and a solvent having a relative dielectric constant of 2.5 or more. It is preferable that the solvent having a relative dielectric constant of less than 2.5 is any one of the solvents mixed so as to exceed 50 parts by volume when the volume of the whole solvent is 100 parts by volume.

  In the separation method having such a configuration, the mobile phase has a solvent having a relative dielectric constant less than a certain value, or a solvent having a relative dielectric constant less than a certain value, and a solvent having a relative dielectric constant greater than a certain value. By using one of the mixed solvents mixed in an appropriate ratio, elution of capsaicinoids and / or capsinoids from the stationary phase can be controlled more appropriately, and separation of normal phase liquid chromatography can be performed. The performance can be further improved.

  In the separation method of the present invention, the mobile phase is preferably one selected from the group consisting of hexane, hexane / ethanol mixed solvent, hexane / methanol mixed solvent, and hexane / acetone mixed solvent.

  In the separation method having such a configuration, elution of capsaicinoids and / or capsinoids from the stationary phase can be more appropriately controlled by using an appropriate solvent or mixed solvent as the mobile phase, and the normal phase The separation performance of liquid chromatography can be further improved.

  In the separation method of the present invention, the mobile phase is preferably hexane or a hexane / ethanol mixed solvent.

  In the separation method having such a configuration, capsaicinoids and / or mixed solvents are used by using hexane or a hexane / ethanol mixed solvent that is the most suitable solvent or mixed solvent and has little influence on the human body as the mobile phase. Alternatively, the elution of capsinoids from the stationary phase can be controlled more appropriately, and the separation performance of normal phase liquid chromatography can be improved most. Further, the capsaicinoids and / or capsinoids can be separated from the separated capsaicinoids. The capsaicinoids and / or capsinoids can be used for food additives and the like without taking any special measures to completely remove the solvent.

In the separation method of the present invention, the specific surface area required by the nitrogen adsorption method of the porous organic polymer particles is in the range of 50 m ² / g or more and 2000 m ² / g or less, and the fine surface property obtained by the nitrogen adsorption method. The pore volume is preferably 0.1 ml / g or more and 3.0 ml / g or less.

  In the separation method having such a configuration, by using porous organic polymer particles having an appropriate specific surface area and pore volume as a stationary phase, the stationary phase exhibits an appropriate holding force, and efficiently produces desired capsaicinoids. And / or capsinoids can be obtained.

  According to the separation method of the present invention, as the stationary phase, by using porous organic polymer particles made of a (meth) acrylic acid ester-based crosslinked (co) polymer that easily retains capsaicinoids and capsinoids, Only the desired capsaicinoids and capsinoids can be efficiently separated from the raw materials containing capsaicinoids and capsinoids, respectively. Therefore, according to the separation method of the present invention, high-efficiency separation of capsaicinoids and / or capsinoids, which was difficult with the prior art, can be performed efficiently and stably even on an industrial scale. .

  The method for separating capsaicinoids from a raw material containing capsaicinoids of the present invention is a method of separating by a liquid chromatography method having a stationary phase and a mobile phase. ) It is characterized by using porous organic polymer particles made of a polymer.

  The method for separating capsinoids from a raw material containing capsinoids according to the present invention is a method of separating by liquid chromatography having a stationary phase and a mobile phase. ) It is characterized by using porous organic polymer particles made of a polymer.

  The method for separating capsaicinoids and capsinoids from a raw material containing capsaicinoids and capsinoids according to the present invention is a method of separating by a liquid chromatography method having a stationary phase and a mobile phase. ) It is characterized by using porous organic polymer particles made of an acrylic ester-based crosslinked (co) polymer.

Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the gist of the present invention.
In the present invention, (meth) acrylic acid refers to acrylic acid and methacrylic acid. The (co) polymer is a polymer obtained by polymerizing one kind of monomer and a copolymer obtained by polymerizing a plurality of kinds of monomers.
TECHNICAL FIELD The present invention relates to a method for separating capsaicinoids and / or capsinoids by liquid chromatography, and a porous material comprising a (meth) acrylate-based crosslinked (co) polymer as a stationary phase of the liquid chromatography. Organic polymer particles are used.
The separation method of the present invention is characterized in that it can efficiently separate capsaicinoids and capsinoids from capsaicinoids and raw materials containing capsinoids. According to the separation method of the present invention, it is possible to efficiently separate capsaicinoids or capsinoids from capsaicinoids or raw materials containing capsinoids, even on an industrial scale. Furthermore, even on an industrial scale, capsaicinoids or capsinoids can be efficiently separated from the same raw material containing capsaicinoids and capsinoids, respectively.
Examples of capsaicinoids and raw materials containing capsinoids include extracts from capsicum, capsicum non-paste-fixed varieties “CH-19 sweet”, and the like by known methods.

In the present invention, “capsaicinoids” are a group of compounds having the same basic skeleton as capsaicin and having the same physiological activity as capsaicin (the above-mentioned energy metabolism promoting action, obesity suppressing action, immunostimulating action, etc.). It is a general term. The “basic skeleton similar to capsaicin” here has a benzene ring and an amide group as shown in the general formula of the following formula (1), and the nitrogen atom of the benzene ring and the amide group is methylene. An alicyclic hydrocarbon group or an aromatic hydrocarbon group (R in the following formula (1)) is added to the carbon atom of the amide group, and one or more on the benzene It is a structure in which an alkoxy group (—OR _a in the following formula (1)) and one or more hydroxy groups are substituted.

（上記式（１）中の置換基Ｒは、炭素数５〜１５の脂肪族炭化水素基、又は炭素数６〜１５の芳香族炭化水素基を示す。ｍ、ｎはそれぞれ１〜４の自然数であり、ｍとｎの和が２以上５以下であれば、ヒドロキシ基及びアルコキシ基（‐ＯＲ_ａ）の置換位置は問わない。アルコキシ基上のＲ_ａは、炭素数１〜３の脂肪族炭化水素基である。）

(The substituent R in the above formula (1) represents an aliphatic hydrocarbon group having 5 to 15 carbon atoms or an aromatic hydrocarbon group having 6 to 15 carbon atoms. M and n are natural numbers of 1 to 4 respectively. If the sum of m and n is 2 or more and 5 or less, the substitution position of the hydroxy group and the alkoxy group (—OR _a ) is not limited, and R _a on the alkoxy group is an aliphatic group having 1 to 3 carbon atoms. It is a hydrocarbon group.)

  Specific examples of capsaicinoids include those having a structure as shown in the following formula (2).

（上記式（２）中の置換基Ｒは、炭素数５〜１５の脂肪族炭化水素基、又は炭素数６〜１５の芳香族炭化水素基を示す。Ｒ_１〜Ｒ_５は、置換基Ｒの例であり、Ｒ_１：カプサイシン、Ｒ_２：ジヒドロカプサイシン、Ｒ_３：ノルジヒドロカプサイシン、Ｒ_４：ホモカプサイシン、Ｒ_５：ホモジヒドロカプサイシンである。）

(The substituent R in the above formula (2) represents an aliphatic hydrocarbon group having 5 to 15 carbon atoms or an aromatic hydrocarbon group having 6 to 15 carbon atoms. R _{1 to} R ₅ are substituent R R _{1 is} capsaicin, R _{2 is} dihydrocapsaicin, R _{3 is} nordihydrocapsaicin, R _{4 is} homocapsaicin, and R ₅ is homodihydrocapsaicin.)

In the present invention, “capsinoids” are a group of compounds having the same basic skeleton as capsiate and having the same physiological activity as capsiate (the aforementioned energy metabolism promoting effect, obesity suppressing effect, immunostimulating effect, etc.). It is a general term. The “basic skeleton similar to capsiate” herein has a benzene ring and an ester group as shown in the general formula of the following formula (3), and the oxygen atom of the benzene ring and the ester group is methylene. An aliphatic hydrocarbon group or an aromatic hydrocarbon group (R in the following formula (3)) is added to the carbon atom of the ester group, and one or more on the benzene It is a structure in which an alkoxy group (—OR _a in the following formula (3)) and one or more hydroxy groups are substituted.

（上記式（３）中の置換基Ｒは、炭素数５〜１５の脂肪族炭化水素基、又は炭素数６〜１５の芳香族炭化水素基を示す。ｍ、ｎはそれぞれ１〜４の自然数であり、ｍとｎの和が２以上５以下であれば、ヒドロキシ基及びアルコキシ基（‐ＯＲ_ａ）の置換位置は問わない。アルコキシ基上のＲ_ａは、炭素数１〜３の脂肪族炭化水素基である。）

(The substituent R in the above formula (3) represents an aliphatic hydrocarbon group having 5 to 15 carbon atoms or an aromatic hydrocarbon group having 6 to 15 carbon atoms. M and n are natural numbers of 1 to 4 respectively. If the sum of m and n is 2 or more and 5 or less, the substitution position of the hydroxy group and the alkoxy group (—OR _a ) is not limited, and R _a on the alkoxy group is an aliphatic group having 1 to 3 carbon atoms. It is a hydrocarbon group.)

  Specific examples of capsinoids include those having a structure as shown in the following formula (4).

（上記式（４）中の置換基Ｒは、炭素数５〜１５の脂肪族炭化水素基、又は炭素数６〜１５の芳香族炭化水素基を示す。Ｒ_６〜Ｒ_８は、置換基Ｒの例であり、Ｒ_６：カプシエイト、Ｒ_７：ジヒドロカプシエイト、Ｒ_８：ノルジヒドロカプシエイトである。）

(The substituent R in the above formula (4) represents an aliphatic hydrocarbon group having 5 to 15 carbon atoms or an aromatic hydrocarbon group having 6 to 15 carbon atoms. R _{6 to} R ₈ are substituent R R _{6 is} capsiate, R _{7 is} dihydrocapsiate, and R _{8 is} nordihydrocapsiate.

The liquid chromatography used in the present invention is preferably normal phase liquid chromatography. Normal phase liquid chromatography is a method in which a mobile phase having a polarity lower than that of the stationary phase is passed through the stationary phase, and a sample is loaded and separated.
The technique of stepwise purification by silica gel chromatography and reverse phase chromatography on silica gel disclosed in Patent Document 3 described above, as pointed out in Patent Document 4 described above, capsinoids are gradually added in the purification process. Therefore, there is a problem that capsinoids cannot be extracted with high yield. One reason for this is that water is contained in the mobile phase used in the reverse phase chromatography performed in Patent Document 3, so that a part of the target product capsinoid is lost by hydrolysis. It is estimated that
When using normal-phase liquid chromatography, unlike in reverse-phase chromatography, water is not essential in the mobile phase, so using anhydrous solvents in the mobile phase hydrolyzes capsaicinoids and capsinoids during the separation. This is suitable for the separation of the raw materials described above.

(Stationary phase)
In the present invention, porous organic polymer particles made of a (meth) acrylic ester-based crosslinked (co) polymer are used as a stationary phase for liquid chromatography. This makes it possible to separate capsaicinoids or capsinoids, as well as to separate capsaicinoids and capsinoids, as described in the Examples below. The porous organic polymer particles made of this (meth) acrylic acid ester-based crosslinked (co) polymer can be used for industrial separation from a small particle size of several μm suitable for high-level separation when used as a stationary phase. It is characterized by excellent production reproducibility of particles having a wide range of particle sizes up to a large particle size of several hundreds μm suitable for the above. As a range of the particle diameter of the porous organic polymer particles, those having a range of 1 μm or more and 2 mm or less can be generally used.

In the present invention, capsaicinoids and solutions containing capsinoids were separated by using porous organic polymer particles made of a (meth) acrylic acid ester-based crosslinked (co) polymer as a stationary phase. In some cases, the capsaicinoids and capsinoids have higher adsorptivity to the stationary phase than conventional silica gel carriers, so that the desired capsaicinoids and / or capsinoids may be obtained from a raw material extracted with a polar organic solvent (alcohol or the like). Even in the case of separation, the extracted solution can be directly injected into the column, and the polar organic solvent as the extraction solvent can be removed from the extract before the column injection, or other solvents other than the polar organic solvent This is extremely advantageous when the present invention is industrially implemented.
The reason why capsaicinoids and capsinoids are highly adsorbed to porous organic polymer particles made of (meth) acrylic acid ester-based crosslinked (co) polymer is that the crosslinked (co) polymer has an ester group. The crosslinked (co) polymer itself has high polarity and high holding power, and capsinoids themselves also have an ester group, and capsaicinoids have an amide group having a structure very similar to that of the ester group. Examples include high affinity between the crosslinked (co) polymer and capsaicinoids or capsinoids. In addition, capsaicinoids and capsinoids can be separated from the same raw material because the affinity of the crosslinked (co) polymer itself for the amide group of the capsaicinoid and the ester group of the capsinoid Is different from sex.

The porous organic polymer particles made of the (meth) acrylic ester-based crosslinked (co) polymer used in the present invention are not particularly limited as long as they can be used for liquid chromatography. Typical (meth) acrylic acid ester-based crosslinked (co) polymers include, for example, copolymers of (meth) acrylic acid esters and derivatives thereof with (meth) acrylic acid polyol esters, (meth) acrylic acid polyols. A homopolymer of ester or at least two types of copolymers of (meth) acrylic acid polyol ester may be mentioned.
Among the examples given above, specific examples of the (meth) acrylic ester-based crosslinked (co) polymer include homopolymers of (meth) acrylic acid polyol esters and (meth) acrylic acid esters and (meth) acrylic acid polyols. Cross-linked copolymers with esters are preferred.
Specific examples of (meth) acrylic acid esters and derivatives thereof include (meth) acrylic acid alkyl esters such as methyl (meth) acrylate and ethyl (meth) acrylate; hydroxyethyl (meth) acrylate, ( Hydroxypropyl (meth) acrylate, 3-chloro-2-hydroxypropyl (meth) acrylate, 2-chloroethyl (meth) acrylate, glycidyl (meth) acrylate, dimethylaminoethyl (meth) acrylate, (meth) acrylic And (meth) acrylic acid ester derivatives such as diethylaminoethyl acid, tetrahydrofurfuryl (meth) acrylate, and 2,3-dihydroxypropyl (meth) acrylate.

(Meth) acrylic acid polyol ester includes (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, glycerin di (meth) acrylate, glycerin tri (meth) acrylate, and trimethylol. Propane tri (meth) acrylate, tetrahydroxybutane di (meth) acrylate, butanediol di (meth) acrylate, neopentyl glycol di (meth) acrylate, hexanediol di (meth) acrylate, pentaerythritol di (meth) acrylate, di Examples include pentaerythritol tetra (meth) acrylate.
These cross-linked (co) polymers and the like can be used as they are, but if necessary, further functional groups may be introduced and used. Examples of functional groups to be introduced include oxygen-containing functional groups such as hydroxyl groups, ester groups, ether groups, and carbonyl groups, nitrogen-containing functional groups such as amino groups, amide groups, nitro groups, and nitrile groups, thiol groups, and sulfone groups. And a sulfur-containing functional group. Among these, a hydroxyl group, an ester group, an amino group, an amide group, and a nitrile group are preferable, and a hydroxyl group and an ester group are more preferable.

  The (meth) acrylic acid ester-based crosslinked (co) polymer used in the present invention has a (meth) acrylic acid ester monomer as a repeating unit at 50 mol% or more, preferably 60 mol% or more, most preferably 70 mol% or more, And what is necessary is just to have a monomer which has an acrylic group like another polyfunctional acrylic ester, or another ethylenically unsaturated bond two or more as a crosslinking component. The crosslinked (co) polymer used in the present invention may contain a monomer other than these monomers as a repeating unit, but the ratio is preferably 5 mol% or less, more preferably 1 mol% or less, Most preferably, it is 0.01 mol% or less.

The degree of cross-linking of the cross-linked (co) polymer is only required to be strong enough to be used as a column for liquid chromatography, and is usually 0.1% by weight or more, preferably 10% by weight or more, and most preferably 20%. % By weight or more. This is because the mechanical strength of the crosslinked (co) polymer cannot be maintained if the degree of crosslinking is less than 0.1% by weight. Here, the degree of crosslinking is a value obtained by dividing the weight of the crosslinked monomer by the sum of the weight of the monomer constituting the main skeleton of the polymer and the weight of the crosslinking monomer.
In addition, the crosslinked (co) polymer illustrated above can be produced by a known method.

In the present invention, the organic polymer particles made of a (meth) acrylic ester-based crosslinked (co) polymer are preferably porous. This is because the use of a porous material is effective for increasing the throughput during separation by normal phase chromatography.
Specifically, the degree of porosity of organic polymer particles (hereinafter sometimes simply referred to as “organic polymer particles”) made of a (meth) acrylate-based crosslinked (co) polymer is nitrogen. The specific surface area determined by the adsorption method is usually 1 m ² / g or more, preferably 50 m ² / g or more, more preferably 100 m ² / g or more, and usually 2000 m ² / g or less.
The pore volume of the organic polymer particles determined by the nitrogen adsorption method is usually 0.01 ml / g or more, preferably 0.1 ml / g or more, more preferably 0.2 ml / g or more, Usually 3.0 ml / g or less.
The specific surface area can be measured using, for example, Flowsorb 2300 (manufactured by Micromeritics), and the pore volume can be measured using, for example, ASAP2400 (manufactured by Micromeritics). .
The average particle size of the organic polymer particles is usually in the range of 1 μm to 2 mm, preferably in the range of 3 μm to 2 mm, and more preferably in the range of 4 μm to 1 mm. Further, the particle shape of the organic polymer particles is preferably true spherical, and the particle size distribution is preferably narrow.

The porous organic polymer particles comprising the (meth) acrylic acid ester-based crosslinked (co) polymer of the present invention can be appropriately selected from commercially available products as long as the above properties are satisfied. Specifically, Diaion HP1MG, HP2MG (pore volume: 1.2 ml / g, specific surface area: 470 m ² / g), MCIGELCH2MGM, CHP2MG, CHP2MGY, etc., Sepabeads SP2MGS, FP-HG, etc. (Mitsubishi Chemical Corporation) Product name), Amberlite XAD7, XAD7HP (specific surface area: 380 m ² / g), amber chrome CG-71 (specific surface area: 500 m ² / g), etc. (above, manufactured by Rohm and Haas; product name) Etc.

  The liquid chromatography used in the present invention is preferably normal phase liquid chromatography. In order to obtain normal phase liquid chromatography, it is necessary to select a mobile phase having a polarity lower than that of the stationary phase. As an index representing polarity, there is a relative dielectric constant. For example, according to Polymer Handbook 3rd edition, polymethyl methacrylate has a relative dielectric constant of 2.57 to 3.6.

(Mobile phase)
As the mobile phase, an organic solvent having a polarity lower than that of the stationary phase described above can be used. However, in order to suppress the reduction in the recovery rate due to the hydrolysis of capsaicinoids and capsinoids, the mobile phase contains water. Preferably not.
Note that the “water-free” solvent herein does not intentionally contain water, but only a relatively small amount of water as an impurity capable of achieving the object of the present invention ( It is a solvent that may be contained in an extremely small amount (particularly preferably), and generally has a water content of about the same as an anhydrous solvent used in liquid chromatography, that is, 1.0% by weight or less, preferably 0.5% by weight or less. It means a solvent containing water. The water content of the mobile phase is more preferably 0.1% by weight or less, and most preferably 0.05% by weight or less.

  More specifically, elution of capsaicinoids and / or capsinoids from the stationary phase can be appropriately controlled, and the mobile phase can be improved from the viewpoint of improving the separation performance of normal phase liquid chromatography. A solvent having a lower relative dielectric constant than the porous organic polymer particles used as the stationary phase, or a solvent having the lower relative dielectric constant and a higher relative dielectric constant than the porous organic polymer particles used as the stationary phase. It is preferable that it is either one of the mixed solvent with the solvent which has a rate.

When the relative permittivity of the mobile phase is defined by the relative dielectric constant, a solvent having a relative dielectric constant of less than 2.5, or a solvent having a relative dielectric constant of less than 2.5 and a solvent having a relative dielectric constant of 2.5 or more. The solvent having a relative dielectric constant of less than 2.5 is more than 50 parts by volume, preferably 70 parts by volume or more, more preferably 80 parts by volume or more when the total volume of the solvent is 100 parts by volume. It is preferable to select any one of the mixed solvents as the mobile phase. This is because a solvent having a relative dielectric constant less than a certain value or a solvent having a relative dielectric constant less than a certain value and a solvent having a relative dielectric constant greater than a certain value are mixed in the mobile phase at an appropriate ratio. By using any one of the mixed solvents, elution of capsaicinoids and / or capsinoids from the stationary phase can be controlled more appropriately, and the separation performance of normal phase liquid chromatography can be further improved. Because it can.
In the present invention, the relative dielectric constant refers to the ratio of the dielectric constant of a substance to the dielectric constant of a vacuum, and the measurement method conforms to JISC2138.
Specific examples of the solvent having a relative dielectric constant of less than 2.5 that can be used as the mobile phase include n-pentane (relative dielectric constant 1.844), n-hexane (relative dielectric constant 1.890), n- Saturated aliphatic hydrocarbons such as heptane (relative permittivity 1.924), n-octane (relative permittivity 1.948), isooctane (relative permittivity 1.943), cyclohexane (relative permittivity 2.052), And one or more hydrocarbon organic solvents selected from the group consisting of aromatic hydrocarbons such as toluene (dielectric constant 2.24) and xylene (dielectric constant 2.266).
These hydrocarbon-based organic solvents may contain a small amount of a polar organic solvent as a solvent having a relative dielectric constant of 2.5 or more, if necessary. This is because it is preferable to use a mixed solvent of a hydrocarbon-based organic solvent and a polar organic solvent because the elution position can be easily adjusted.

  As a polar organic solvent that can be added to the hydrocarbon organic solvent as a solvent having a relative dielectric constant of less than 2.5, the relative dielectric constant is 2.5 or more, and preferably the relative dielectric constant is usually 4 to 4. Selected from 38 organic solvents. Specifically, carbon such as methanol (relative permittivity 33.1), ethanol (relative permittivity 23.8), n-propanol (relative permittivity 22.2), 2-propanol (relative permittivity 18.3), etc. 1 to 6 aliphatic alcohols; alkyl acetates having 1 to 6 carbon atoms in the alkyl chain such as ethyl acetate (dielectric constant 6.02) and propyl acetate (dielectric constant 6.002); chloroform ( Chlorinated organic compounds such as dielectric constant 4.9) and dichloromethane (dielectric constant 9.1); alkyl ketones such as acetone (dielectric constant 20.7) and methyl ethyl ketone (dielectric constant 18.51); Alkyl ethers such as diethyl ether (dielectric constant 4.197), diisopropyl ether (dielectric constant 4.49), tetrahydrofuran (dielectric constant 7.58), and acetonitrile (dielectric constant) 37.5) Any one or two or more organic solvent selected may be mentioned from the group consisting like.

  The mobile phase in the liquid chromatography method is preferably one selected from the group consisting of hexane, hexane / ethanol mixed solvent, hexane / methanol mixed solvent, and hexane / acetone mixed solvent. This is because the elution of capsaicinoids and / or capsinoids from the stationary phase can be controlled more appropriately by using an appropriate solvent or mixed solvent for the mobile phase, and the performance of separation in normal phase liquid chromatography can be controlled. It is because it can improve further.

  It is particularly preferable that the mobile phase is hexane or a hexane / ethanol mixed solvent. This is because the mobile phase is the most suitable solvent or mixed solvent and has little influence on the human body, etc., so that the capsaicinoids and / or capsinoids stationary phase can be obtained by using hexane or a hexane / ethanol mixed solvent. In order to better control the separation performance of normal phase liquid chromatography, and to completely remove the solvent from the separated capsaicinoids and / or capsinoids. This is because the capsaicinoids and / or capsinoids can be used for food additives and the like without taking any special measures.

  The amount of the polar organic solvent that can be contained in the hydrocarbon-based organic solvent varies depending on the polarity of the stationary phase, the type of the organic solvent, the relative dielectric constant, etc., and cannot be determined uniformly. Is usually less than 50 parts by volume, preferably 30 parts by volume or less, more preferably 20 parts by volume or less.

(Chromatography equipment)
As the apparatus for performing the liquid chromatography separation performed in the present invention, known apparatuses such as a high performance liquid chromatography (HPLC) apparatus, an intermediate pressure liquid chromatography apparatus, and a low pressure liquid chromatography apparatus can be used.
Further, in the normal phase liquid chromatography separation performed in the present invention, a single column method, a multistage column method, a valve in which a plurality of columns are circulated and connected, and mobile phase flow, sample addition, and separation component extraction are provided between the columns. A so-called simulated moving bed (simulated mobile phase, Simulated Moving Bed or SMB) method, an improved simulated moving bed (ISMB) method, and the like are possible.

  According to the separation method of the present invention, as the stationary phase, by using porous organic polymer particles made of a (meth) acrylic acid ester-based crosslinked (co) polymer that easily retains capsaicinoids and capsinoids, Only the desired capsaicinoids and capsinoids can be efficiently separated from the raw materials containing capsaicinoids and capsinoids, respectively. Therefore, according to the separation method of the present invention, high-efficiency separation of capsaicinoids and / or capsinoids, which was difficult with the prior art, can be performed efficiently and stably even on an industrial scale. .

  Specific embodiments of the present invention will be described below in more detail with reference to examples. However, the present invention is not limited to these examples unless it exceeds the gist.

Example 1
Using a high-performance liquid chromatography (HPLC) apparatus equipped with a liquid feed pump, an auto sample injector, an ultraviolet absorbance detector, and a data processor, the stationary phase is from a (meth) acrylic ester-based crosslinked (co) polymer. A porous organic polymer particle, MCIGEL CHP2MG (Mitsubishi Chemical Corporation, particle diameter 10 μm) packed in a stainless steel column having an inner diameter of 4.6 mm and a length of 250 mm was used. Under normal phase liquid chromatography conditions using n-hexane / ethanol = 87.5 / 12.5 (volume ratio) as a mobile phase, liquid was passed through the liquid feed pump at a flow rate of 1.00 ml / min. The temperature in the laboratory was 25 ° C.

As a sample (raw material to be separated), a solution in which 1 mg of capsaicin (manufactured by Wako Pure Chemical Industries, for biochemistry) was dissolved in 1.00 g of ethanol (manufactured by Junsei Chemical Co., Ltd., reagent special grade) was used, and 20 μl was placed in a column with an auto sample injector. Poured and separated.
The chromatogram obtained by setting the wavelength of the ultraviolet absorbance detector to 280 nm is shown in FIG. In FIG. 1, the horizontal axis represents a value obtained by dividing the retention volume (the value obtained by multiplying the retention time by the flow rate) by the column volume, and the vertical axis represents the absorbance at a wavelength of 280 nm. The same applies to FIGS. 2 to 13 described later. With capsaicin alone, a capsaicin peak was sharply detected at a position where (retention volume) / (column volume) was about 4.1.

(Example 2)
The same conditions as in Example 1 were used except that 1 mg of dihydrocapsaicin (manufactured by Sigma, purity 90% or more) dissolved in 1.00 g of ethanol (manufactured by Junsei Chemical, reagent grade) was used as a sample. Normal phase chromatographic separation was performed.
The obtained chromatogram is shown in FIG. With dihydrocapsaicin alone, a peak of dihydrocapsaicin was detected sharply at a position where (retention volume) / (column volume) was about 3.7. In comparison with FIG. 1, it can be seen that capsaicin and dihydrocapsaicin can be separated from each other because the position of (retention volume) / (column volume) is distant and the peak is sharp.

(Example 3)
Normal phase chromatography under the same conditions as in Example 1 except that 1.00 g of pepper powder (manufactured by Takasago Pharmaceutical Co., Ltd.) was extracted with 25 ml of ethanol (manufactured by Junsei Chemical, reagent grade) as a sample. Separation was performed.
The obtained chromatogram is shown in FIG. In FIG. 3, there is a capsaicin-derived peak at substantially the same (retained volume) / (column volume) as in FIG. 1, and a dihydrocapsaicin-derived peak at approximately the same (retained volume) / (column volume) as in FIG. You can see that it exists.
Therefore, in this Example, it can be seen that capsaicin and dihydrocapsaicin can be separated from the pepper extract ethanol extract.

Example 4
The sample was the same as in Example 1 except that 1.00 g of pepper powder (manufactured by Takasago Pharmaceutical Co., Ltd.) was extracted with 25 ml of ethanol (manufactured by Junsei Chemical, reagent grade) and the injection volume was 40 μl. Normal phase chromatographic separation was performed under the conditions.
The obtained chromatogram is shown in FIG. When porous organic polymer particles comprising a (meth) acrylic acid ester-based crosslinked (co) polymer were used for the stationary phase, as can be seen from comparison with Example 3 in which the injection amount was 20 μl for the same sample, Even in the case of FIG. 4 where the injection amount of the powdered ethanol extract is doubled, the influence of ethanol as the extractant is small, and there is a peak derived from capsaicin at the same (retention volume) / (column volume) as in FIG. It can be seen that a peak derived from dihydrocapsaicin is present at substantially the same (retention volume) / (column volume) as in FIG.

(Example 5)
As a sample, Capsiate Natura (a food containing chili extract from Ajinomoto Co., Inc. containing a chili extract and rapeseed oil as a content in a capsule consisting of starch, glycerin, carrageenan and a pH adjuster, in 0.33 g of 1 capsule Normal phase chromatographic separation was performed under the same conditions as in Example 1, except that the content in 1 grain (containing 1.0 mg of capsiate) was dissolved in 10.0 g of hexane.
The obtained chromatogram is shown in FIG. In FIG. 5, it can be seen that there are two peaks at positions where (retention volume) / (column volume) is smaller than capsaicin and dihydrocapsaicin. These two peaks indicate the area where (retention volume) / (column volume) is at a position of about 2.7, based on the abundance ratio of isolated capsiate and dihydrocapsiate described in Non-Patent Document 1. It can be determined that the peak with a small value is dihydrocapsiate, and the peak with a large area value existing at a position where (retention volume) / (column volume) is about 3.0 is capsiate.
From this, it can be seen that capsiate and dihydrocapsiate can be separated from the chili extract. Furthermore, the values of (retention volume) / (column volume) of the capsiate and dihydrocapsiate obtained in this example are (retention volume) / (column volume) of capsaicin and dihydrocapsaicin shown in Examples 1 to 3, respectively. It can be seen that capsiate and dihydrocapsiate can be separated from capsaicin and dihydrocapsaicin.

(Example 6)
Using a high-performance liquid chromatography (HPLC) apparatus equipped with a liquid feed pump, an auto sample injector, an ultraviolet absorbance detector, and a data processor, the stationary phase is from a (meth) acrylic ester-based crosslinked (co) polymer. A porous organic polymer particle, MCIGEL CHP2MG (Mitsubishi Chemical Corporation, particle diameter 10 μm) packed in a stainless steel column having an inner diameter of 4.6 mm and a length of 250 mm was used. Under normal phase liquid chromatography conditions using n-hexane / ethanol = 90/10 (volume ratio) as a mobile phase, liquid was passed through the liquid feed pump at a flow rate of 1.00 ml / min. The temperature in the laboratory was 25 ° C.

As a sample, Capsiate Natura (a food containing chili extract from Ajinomoto Co., Inc. containing a chili extract and rapeseed oil as a content in a capsule consisting of starch, glycerin, carrageenan and a pH adjuster, in 0.33 g of 1 capsule The contents in one grain (containing 1.0 mg of capsiate) were used as they were without being dissolved in hexane, and 60 μl was injected into the column with an autosample injector and separated.
The obtained chromatogram is shown in FIG. It can be seen that even when the amount of load increases as compared with Example 5, it is possible to separate capsiate and dihydrocapsiate.

(Example 7)
A high-performance liquid chromatography (HPLC) apparatus equipped with a liquid feed pump, a manual sample injector, an ultraviolet absorbance detector, and a data processing apparatus is used, and the stationary phase is composed of a (meth) acrylate-based crosslinked (co) polymer. Sepa beads SP2MGS (particle diameter: 145 μm, manufactured by Mitsubishi Chemical Corporation), which are porous organic polymer particles, packed in a stainless steel column having an inner diameter of 20 mm and a length of 500 mm were used. Under normal phase liquid chromatography conditions using n-hexane / ethanol = 90/10 (volume ratio) as a mobile phase, the solution was fed at a flow rate of 2.36 ml / min. The temperature in the laboratory was 25 ° C.

As a sample, Capsiate Natura (a food containing chili extract from Ajinomoto Co., Inc. containing a chili extract and rapeseed oil as a content in a capsule consisting of starch, glycerin, carrageenan and a pH adjuster, in 0.33 g of 1 capsule The contents in 8 grains (containing 1.0 mg of capsiate) were used as they were, and 1.2 ml was injected into the column with a manual sample injector, and separation was performed.
FIG. 7 shows a chromatogram obtained by setting the wavelength of the ultraviolet absorbance detector to 280 nm. In FIG. 7, there are peaks derived from capsiate and dihydrocapsiate at substantially the same (retention volume) / (column volume) as in FIG.
Therefore, it can be seen that in this example, it is possible to separate the capsiate and dihydrocapsiate.

(Comparative Example 1)
The same HPLC apparatus used in Example 1 was used, and the stationary phase was MCIGEL CHP5C (Mitsubishi Chemical Corporation, particle size 10 μm), which is a porous organic polymer particle made of a polystyrene-based crosslinked (co) polymer. ) Was packed in a stainless steel column having an inner diameter of 4.6 mm and a length of 150 mm. Under normal phase liquid chromatography conditions using n-hexane / ethanol = 98/2 (volume ratio) as a mobile phase, liquid was passed through a liquid feed pump at a flow rate of 1.00 ml / min. The temperature in the laboratory was 25 ° C.

Using a solution prepared by dissolving 1 mg of capsaicin (manufactured by Wako Pure Chemical Industries, Ltd., for biochemistry) in 1.00 g of ethanol (manufactured by Junsei Chemical, special grade for reagent), 20 μl was injected into the column with an autosample injector and separated.
The chromatogram obtained by setting the wavelength of the ultraviolet absorbance detector to 280 nm is shown in FIG. Unlike Example 1, it can be seen that a clear peak of capsaicin cannot be confirmed. Therefore, when porous organic polymer particles made of polystyrene-based crosslinked (co) polymer are used as the stationary phase, porous organic polymer particles made of (meth) acrylate-based crosslinked (co) polymer are It is understood that capsaicin cannot be separated unlike the case of using (Example 1).

(Comparative Example 2)
The same conditions as in Comparative Example 1 were used except that 1 mg of dihydrocapsaicin (manufactured by Sigma, purity 90% or higher) dissolved in 1.00 g of ethanol (manufactured by Junsei Chemical, reagent grade) was used as a sample. Normal phase chromatographic separation was performed.
The obtained chromatogram is shown in FIG. Also in this case, a clear peak of dihydrocapsaicin cannot be confirmed, and it can be seen that separation from capsaicin has not been achieved. Therefore, when porous organic polymer particles made of polystyrene-based crosslinked (co) polymer are used as the stationary phase, porous organic polymer particles made of (meth) acrylate-based crosslinked (co) polymer are It can be seen that dihydrocapsaicin cannot be separated unlike when used (Example 2).

(Reference Example 1)
The same HPLC apparatus as used in Example 1 was used, and as a stationary phase, a silica gel packed column YMC Pack SIL SL12S11-2546WT (particle diameter 10 μm; column inner diameter 4.6 mm, column length 250 mm, manufactured by YMC) ) Was used. Under normal phase liquid chromatography conditions using n-hexane / ethanol = 95/5 (volume ratio) as a mobile phase, liquid was passed through the liquid feed pump at a flow rate of 1.00 ml / min. The temperature in the laboratory was 25 ° C.

Using a solution prepared by dissolving 1 mg of capsaicin (manufactured by Wako Pure Chemical Industries, Ltd., for biochemistry) in 1.00 g of ethanol (manufactured by Junsei Chemical, special grade for reagent), 20 μl was injected into the column with an autosample injector and separated.
The chromatogram obtained by setting the wavelength of the ultraviolet absorbance detector to 280 nm is shown in FIG. With capsaicin alone, a capsaicin peak was detected slightly broadly at a position where (retention volume) / (column volume) was about 5.2.

(Reference Example 2)
The same conditions as in Reference Example 1 were used except that 1 mg of dihydrocapsaicin (manufactured by Sigma, purity 90% or more) dissolved in 1.00 g of ethanol (manufactured by Junsei Chemical, reagent grade) was used as a sample. Normal phase chromatographic separation was performed.
The obtained chromatogram is shown in FIG. In the case of dihydrocapsaicin alone, a dihydrocapsaicin peak was detected slightly broadly at a position where (retention volume) / (column volume) was about 4.9.

(Comparative Example 3)
Normal phase chromatography under the same conditions as in Reference Example 1 except that 1.00 g of pepper powder (manufactured by Takasago Pharmaceutical Co., Ltd.) was extracted with 25 ml of ethanol (manufactured by Junsei Chemical Co., Ltd., reagent grade) as a sample. Separation was performed.
The obtained chromatogram is shown in FIG. Although a broad peak exists at a position where (retention volume) / (column volume) is about 5.0, it is understood that capsaicin and dihydrocapsaicin cannot be separated from the pepper extract ethanol extract.
In this Comparative Example 3 in which a sample containing a large amount of alcohol was injected into a silica gel packed column, the separation ability for separating capsaicin and dihydrocapsaicin was very poor. Therefore, when using a column packed with silica gel, it is inappropriate to inject the sample extracted with alcohol directly into the column, either to remove alcohol from the extract before column injection or to use other solvents other than alcohol. It is necessary to extract using

(Comparative Example 4)
The sample was the same as in Reference Example 1 except that 1.00 g of red pepper powder (manufactured by Takasago Pharmaceutical Co., Ltd.) was extracted with 25 ml of ethanol (manufactured by Junsei Chemical Co., Ltd., reagent grade) and the injection volume was 40 μl. Normal phase chromatographic separation was performed under the conditions.
The obtained chromatogram is shown in FIG. When silica gel is used for the stationary phase, increasing the injection amount of the pepper extract ethanol extract not only lowers the resolution of capsaicin and dihydrocapsaicin due to the effect of ethanol as the extractant, but also (retention volume) / ( It can be seen that the column volume) is about 4.9 and the retention of capsaicinoids is shortened.

  The present invention can be used in capsaicinoids and any field that separates capsinoids, and capsaicinoids and capsinoids separated by the method of the present invention are useful for, for example, foods, pharmaceuticals, quasi drugs, cosmetics, etc. It is.

The chromatogram obtained as a result of Example 1 of this invention is shown. The chromatogram obtained as a result of Example 2 of this invention is shown. The chromatogram obtained as a result of Example 3 of this invention is shown. The chromatogram obtained as a result of Example 4 of this invention is shown. The chromatogram obtained as a result of Example 5 of this invention is shown. The chromatogram obtained as a result of Example 6 of this invention is shown. The chromatogram obtained as a result of Example 7 of this invention is shown. The chromatogram obtained as a result of the comparative example 1 of this invention is shown. The chromatogram obtained as a result of the comparative example 2 of this invention is shown. The chromatogram obtained as a result of Reference Example 1 of the present invention is shown. The chromatogram obtained as a result of Reference Example 2 of the present invention is shown. The chromatogram obtained as a result of the comparative example 3 of this invention is shown. The chromatogram obtained as a result of the comparative example 4 of this invention is shown.

Claims (9)

In a method of separating by a liquid chromatography method having a stationary phase and a mobile phase,
A method for separating capsaicinoids from a raw material containing capsaicinoids, characterized in that porous organic polymer particles comprising a (meth) acrylic acid ester-based crosslinked (co) polymer are used as the stationary phase. In a method of separating by a liquid chromatography method having a stationary phase and a mobile phase,
A method for separating capsinoids from a raw material containing capsinoids, characterized in that porous organic polymer particles comprising a (meth) acrylic acid ester-based crosslinked (co) polymer are used as the stationary phase. In a method of separating by a liquid chromatography method having a stationary phase and a mobile phase,
Capsaicinoids and capsinoids from raw materials containing capsaicinoids and capsinoids, characterized in that porous organic polymer particles comprising (meth) acrylic acid ester-based crosslinked (co) polymers are used as the stationary phase. How to isolate.   The separation method according to any one of claims 1 to 3, wherein the mobile phase does not contain water.   The mobile phase has a solvent having a lower relative dielectric constant than the porous organic polymer particles, or a solvent having the lower relative dielectric constant and a higher relative dielectric constant than the porous organic polymer particles. The separation method according to any one of claims 1 to 4, wherein the separation method is any one of a mixed solvent with a solvent.   The mobile phase is a solvent having a relative dielectric constant of less than 2.5, or a solvent having a relative dielectric constant of less than 2.5 and a solvent having a relative dielectric constant of 2.5 or more. 6. The solvent according to claim 1, wherein the solvent is less than 50 parts by volume when the total volume of the solvent is 100 parts by volume. Separation method.   The mobile phase is one selected from the group consisting of hexane, a hexane / ethanol mixed solvent, a hexane / methanol mixed solvent, and a hexane / acetone mixed solvent. The separation method according to item.   The separation method according to any one of claims 1 to 7, wherein the mobile phase is hexane or a hexane / ethanol mixed solvent. The porous specific surface area determined by nitrogen adsorption method of the organic polymer particles, ^{50 m} 2 / g or more and the range 2000 m ² / g, and pore volume determined by nitrogen adsorption method is 0.1 ml / The separation method according to claim 1, wherein the separation method is g or more and 3.0 ml / g or less.

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