JP2003292489A - Method for separating tocopherol compounds and tocotrienol compounds - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for industrially efficiently and stably separating a tocopherol compound or a tocotrienol compound, and to provide a method for separating each homologue of the tocopherol compound and the tocotrienol compound. <P>SOLUTION: This method for separating the desired tocopherol compound or tocotrienol compound from a solution containing the tocopherol compound and the tocotrienol compound. And further the method for separating each homologue (α, β, γ, δ-) of the tocopherol compound and the tocotrienol compound is characterized by using porous organic polymer particles [for example, a polyvinylbenzene-based cross-linked (co)polymer, (meth)acrylate-based cross- linked (co)polymer] as the solid phase of normal phase liquid chromatography. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for separating tocopherols and tocotrienols using normal phase liquid chromatography. More specifically, the present invention relates to a method for separating tocopherol homologues from a solution containing tocopherols and tocotrienols by normal phase liquid chromatography, and simultaneously separating tocopherols and tocotrienols.

[0002]

BACKGROUND OF THE INVENTION Tocopherols and tocotrienols are contained in the deodorizing distillate produced as a by-product in the deodorizing process of soybean oil, rapeseed oil, cottonseed oil, safflower oil, rice bran oil, corn oil, sunflower oil, etc. d -Α, d-β, d
-Γ, d-δ-tocopherol and d-α, d-β,
There are homologues of d-γ, d-δ-tocotrienol. Note that naturally-occurring tocopherols and tocotrienols are all d-forms, but synthetically-derived tocopherols and tocotrienols are usually dl-forms (unless otherwise specified in the present specification, tocopherols and tocotrienols are d-forms, l-forms and d-forms). Both the body and the 1-body mixture are included).

Since tocopherols and tocotrienols have physiological activity as a vitamin E group and have an antioxidant action, they are used in medicines, cosmetics, foods and the like. In recent years, the vitamin E bioactivity is α
-Although inferior to tocopherol, β, γ, δ-tocopherol having an antioxidant effect, δ-tocopherol has an excellent antioxidant effect, and γ-tocopherol has an effect on urination and skin health For example, the functionality of individual tocopherol homologues has been attracting attention, and a method for efficiently isolating them has been required. Further, tocotrienols have also been found to have functions such as hyperlipidemia improving action, atherosclerosis improving action, and anticancer action, and an efficient method for separating them from tocopherols has been demanded. .

However, conventionally, a method such as a molecular distillation method or an ion exchange resin treatment has been used for purifying and concentrating the tocopherols. However, the molecular distillation method makes it difficult to carry out a separation operation, and a high-concentration concentrate is used. It is very difficult to obtain a high yield, and in the ion-exchange resin treatment method, there is a drawback that regeneration of the resin is necessary and complicated steps such as a removal treatment of an eluent that occurs when performing solvent replacement are required. It was difficult to isolate each homologue of tocopherols and to efficiently separate them from tocotrienols. Various methods have been proposed to solve these problems. One of the methods is a method using an organic polymer that is not an ion exchange resin. For example, in JP-B-60-37110, crude tocopherol is applied to a column filled with a styrenedivinylbenzene copolymer porous polymer, and an alcohol solvent is used. A method for obtaining purified tocopherol by developing and eluting in a system is disclosed. This method is a reversed-phase chromatographic separation due to the high polarity of the alcohol solvent system with respect to the styrene-divinylbenzene copolymer porous polymer. In this method, α-tocopherol, (β-tocopherol + γ-tocopherol), δ- Separation between the homologues of tocopherol has not been achieved, and the possibility of separating tocotrienols has not been mentioned at all.

Further, Japanese Patent No. 2757372 discloses that
Crosslinking having multiple vinyl groups in one molecule (60% by weight or more) obtained by aqueous suspension polymerization in the presence of a diluent consisting of an organic solvent that is incompatible with water and compatible with the raw material polymerizable monomer Deodorizing distillation to a column filled with beads for separating and concentrating tocopherols, which is made of a porous polymer containing a binding monomer and has a particle size of 10 μm or more and a gel water content of 30% or more. A method is disclosed in which a developing solvent is allowed to pass through after injecting the product, and tocopherols are separated and concentrated by liquid chromatography. In this method, since methanol, which is recommended as a developing solvent and used specifically, has high polarity with respect to the porous polymer, it is a reverse phase chromatographic separation, and mutual separation of tocopherol homologues, that is, α- Tocopherol, (β-tocopherol +
(γ-tocopherol) and δ-tocopherol cannot be separated, and the possibility of separating tocotrienols is not mentioned.

On the other hand, a method using a silica gel type filler has also been investigated. For example, in Japanese Patent Laid-Open No. 8-59647, a method of separating and purifying a tocopherol component by using a simulated moving bed filled with a silica gel type filler is proposed. Although disclosed, this method does not mention the separability of tocopherol and tocotrienol. Further, JP 2001-122869 A discloses a method for isolating tocotrienols, tocopherols and their isomers from a crude initial raw material by using reverse phase liquid chromatography. As the mobile phase, a solvent containing alcohol and water of a predetermined amount or less is used, and when water enters the mobile phase, the solubility of tocopherols and tocotrienols generally decreases, so a high concentration load cannot be applied and it is industrially impossible. Is disadvantageous to

Further, Japanese Patent Laid-Open No. 2001-261671 discloses a method of chromatographically isolating a vitamin E isomer from a compound containing vitamin E in a supercritical fluid environment using silica gel and reversed phase C18 silica gel. Although disclosed, this method has an industrial drawback that the equipment cost is high because a supercritical fluid is used. In addition, for silica gel-based fillers, it is difficult to control the mixing degree of various metals in the production of silica gel, there is a problem of reproducibility of separation behavior due to the influence of these mixed metal ions, and retention due to the influence of water content. There is a large variation in behavior, and there is a problem with operational stability. Further, even when using a silica gel-based filler having an alkyl group such as C18 group, it is difficult to control the introduction rate and the end cap of the alkyl group-unintroduced portion, and there is a problem in reproducibility of separation behavior. As described above, it has been difficult in the prior art to separate tocopherols and tocotrienols by an industrially efficient and stable method.

[0008]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and an object thereof is industrially efficient and stable tocopherols, a method for separating tocotrienols, and further tocopherols. It is intended to provide a method for separating each homologue and tocotrienols.

[0009]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that the stationary phase has excellent production reproducibility and excellent chemical resistance against acids and alkalis. Separation of tocopherols and tocotrienols, as well as isolation between each homologue of tocopherols, can be performed efficiently and stably by applying normal phase liquid chromatography using porous organic polymer particles Then, they have completed the present invention. That is, the gist of the present invention is by normal phase liquid chromatography from a solution containing tocopherols and tocotrienols,
In the method of separating desired tocopherols, or tocotrienols, further, in the method of separating each homologue of tocopherols and tocotrienols, it is possible to use porous organic polymer particles as a stationary phase of normal phase liquid chromatography. It lies in the characteristic separation method.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method for separating tocopherols and tocotrienols by normal phase chromatography, and it is essential to use porous organic polymer particles as a stationary phase in this normal phase chromatography. . The porous organic polymer particles used in the present invention can be selected from those used in liquid phase chromatography. Typical examples are porous polyvinylbenzene cross-linked (co) polymers and (Meta)
Examples thereof include acrylic acid ester-based crosslinked (co) polymers. As the polyvinylbenzene-based crosslinked (co) polymer,
Styrene, methylstyrene, ethylstyrene, α-methylstyrene, chlorostyrene, chloromethylstyrene,
A cross-linked copolymer of a styrene-based monomer such as p-hydroxystyrene and pt-butoxystyrene and a polyvinylbenzene-based monomer such as divinylbenzene and trivinylbenzene as a crosslinking agent, or divinylbenzene and trivinyl Examples thereof include homopolymers of polyvinylbenzene monomers such as benzene and copolymers composed of two or more kinds. Among these, a styrene-divinylbenzene crosslinking copolymer is particularly preferable. These crosslinked (co) polymers can be produced by a known method.

(Meth) acrylic acid ester-based cross-linking (co)
Examples of the polymer include (meth) acrylic acid esters and their derivatives, and (co) polymers of monomers such as (meth) acrylic acid polyol esters, such as (meth) acrylic acid esters and their derivatives and (meth) acrylic acid esters. ) Copolymer with acrylic acid polyol ester, or homopolymer of (meth) acrylic acid polyol ester or at least 2
There are several kinds of copolymers, and these crosslinked (co) polymers are produced by a known method. Specific examples of the (meth) acrylic acid ester and its derivative include alkyl (meth) acrylates such as methyl (meth) acrylate and ethyl (meth) acrylate; hydroxyethyl (meth) acrylate, (meth) Hydroxypropyl acrylate, 3-chloro-2-hydroxypropyl (meth) acrylate, 2-chloroethyl (meth) acrylate, glycidyl (meth) acrylate, dimethylaminoethyl (meth) acrylate, diethylamino (meth) acrylate Ethyl, tetrahydrofurfuryl (meth) acrylate,
Examples thereof include (meth) acrylic acid ester derivatives such as 2,3-dihydroxypropyl (meth) acrylic acid.

Further, as (meth) acrylic acid polyol ester, (poly) ethylene glycol di (meth) is used.
Acrylate, (poly) propylene glycol di (meth) acrylate, glycerin di (meth) acrylate, glycerin tri (meth) acrylate, trimethylolpropane tri (meth) acrylate, tetrahydroxybutane di (meth) acrylate, butanediol di (meth) ) Acrylate, neopentyl glycol di (meth) acrylate, hexanediol di (meth) acrylate, pentaerythritol di (meth) acrylate, dipentaerythritol tetra (meth) acrylate and the like. These crosslinked (co) polymers and the like can be used as they are, but may be further introduced with a functional group if necessary. As the functional group to be introduced, for example, a hydroxyl group, an ester group, an ether group, an oxygen-containing functional group such as a carbonyl group, an amino group, an amide group, a nitro group, a nitrogen-containing functional group such as a nitrile group, a thiol group, a sulfone group, etc. Examples of the sulfur-containing functional group include, but are preferably a hydroxyl group, an ester group, an amino group, an amide group and a nitrile group, and more preferably a hydroxyl group and an ester group. A preferable specific example of the cross-linked copolymer is a (meth) acrylic acid ester-based cross-linked (co) polymer which is a cross-linked copolymer of a (meth) acrylic acid ester and a (meth) acrylic acid polyol ester, p. Examples include hydrolysates of cross-linked copolymers of -t-butoxystyrene and divinylbenzene,
Particularly preferred is a (meth) acrylic acid ester-based crosslinked (co) polymer.

The fact that the porous organic polymer particles are porous is effective for increasing the amount of treatment at the time of separation by normal phase chromatography. Specifically, the specific surface area determined by the nitrogen adsorption method is 1 m ² / G or more, preferably 50 m ²
/ G or more, more preferably 100 m ² / g or more,
It is 2000 m ² / g or less. The pore volume is 0.01 ml / g or more, preferably 0.1 ml / g or more, more preferably 0.2 ml / g or more, and 3.0
It is less than or equal to ml / g. The average particle size of the porous organic polymer particles is in the range of 1 μm to 2 mm, preferably 3 μm.
m to 2 mm, industrially more preferably 4 μm to 1 mm, and the particle shape is spherical.
The particle size distribution is preferably narrow.

These porous organic polymer particles can be appropriately selected from commercial products as long as they satisfy the above-mentioned porous characteristics. Specific examples of the styrene-divinylbenzene cross-linked copolymer include Diaion HP20 and HP20.
S, HP20SS, HP21, Sepa beads SP20S
S, SP20DF, SP207, SP70, SP70
0, SP825, SP850, MCI GEL CHP
10M, CHP5C, CHP20P, CHP20A, C
HP20Y, CHP55A, CHP55Y, etc. (above, Mitsubishi Chemical Co., Ltd .; trade name); Amberlite XAD2
(Supelco; product name), Amberlite XAD
4, XAD8, XAD16, XAD1180, XAD1
600, XAD2000, XAD2010, Amber Chrome CG-161, CG-300, CG-1000 and the like (above, manufactured by Rohm and Haas Co .; trade name), Dowex L285, L323, L493, V493,
V502 etc. (above, Dow Chemical Co .; trade name), P
LRP-S (manufactured by Polymer Laboratory Co .; trade name),
PRP-1, PRP-3, etc. (manufactured by Hamilton Co .; trade name), SOURCE RPC, RESOURCE RP
C, etc. (manufactured by Amersham Pharmacia Biotech Co .; trade name) and the like.

(Meth) acrylic acid ester crosslinking (co)
As the polymer, Diaion HP1MG, HP2M
G etc., MCI GEL CHP2MGM, CHP2M
G, CHP2MGF, CHP2MGA, CHP2MGY
Etc., Sepa beads FP-HG etc. (all manufactured by Mitsubishi Chemical Co .; trade name), Amberlite XAD7, XAD7HP, Amber Chrome CG-71 etc. (above, Rohm and
Haas Co .; trade name) and the like.

The normal phase liquid chromatography used in the present invention is a method in which a mobile phase having a low polarity is passed through a stationary phase to load and separate a sample. As the mobile phase, an organic solvent having a lower polarity than that of the stationary phase is used and is usually selected from organic solvents having a relative dielectric constant of 2.5 to 1.5. Specifically, n-pentane (relative permittivity 1.844), n-hexane (relative permittivity 1.890), n-heptane (relative permittivity 1.924),
Saturated aliphatic hydrocarbons such as n-octane (relative permittivity 1.948), isooctane (relative permittivity 1.943), cyclohexane (relative permittivity 2.052), and toluene (relative permittivity 2.2
4) and one or more hydrocarbon-based organic solvents selected from the group consisting of aromatic hydrocarbons such as xylene (relative permittivity 2.266). These organic solvents may contain a small amount of polar organic solvents as needed.

The polar organic solvent which can be added is usually selected from organic solvents having a relative dielectric constant of 4 to 38, specifically, methanol (relative dielectric constant 33.1), ethanol (relative dielectric constant 23.
8), n-propanol (relative permittivity 22.2), 2-propanol (relative permittivity 18.3) and other aliphatic alcohols having 1 to 6 carbon atoms; ethyl acetate (relative permittivity 6.02), propyl acetate (relative permittivity) 6.002) etc., alkyl acetates having 1 to 6 carbon atoms in the alkyl chain; chloroform (dielectric constant
4.9), chlorine-based organic compounds such as dichloromethane (dielectric constant 9.1); alkylketones such as acetone (dielectric constant 20.7), methyl ethyl ketone (dielectric constant 18.51); diethyl ether (dielectric constant 4.197), diisopropyl ether (Relative permittivity 4.49), tetrahydrofuran (relative permittivity 7.
58) and the like, and one or more kinds of organic solvents arbitrarily selected from the group consisting of alkyl ethers such as 58) and acetonitrile (relative dielectric constant 37.5). It is preferable to use a mixed solvent of a hydrocarbon organic solvent and these polar organic solvents because the elution position can be easily adjusted. Among these, hexane or a hexane-ethanol mixed solvent, which can be used as a food additive, is preferable in consideration of food use. The amount of polar organic solvent that the hydrocarbon-based organic solvent may contain varies depending on the polarity of the stationary phase, the type of organic solvent, the relative dielectric constant, etc., and cannot be uniformly determined, but is usually less than 50% (volume ratio). It is preferably 40% (volume ratio) or less, more preferably 20% (volume ratio) or less.

The feature of the present invention resides in that it is possible to efficiently separate tocopherols and tocotrienols. Usually, when a solution containing tocopherols and tocotrienols is separated by normal phase liquid chromatography using silica gel as a stationary phase, α-tocopherol and α-tocotrienol; (β-tocopherol + γ-tocopherol) And (β-tocotrienol + γ-tocotrienol); δ
-Tocopherol and δ-tocotrienol are held close to each other, so for example α-tocopherol and β
-Α-tocotrienol elutes between tocopherols, and (β-tocopherol + γ-tocopherol)
And δ-tocopherol (β-tocotrienol +
(γ-tocotrienol) may elute. Therefore, when a silica gel-based filler exhibiting such a retention behavior is used, it is necessary to finely separate each component to separate tocopherols and tocotrienols, which is not industrially efficient. On the other hand, in the present invention, since the retention of the tocotrienols is far apart from the retention of the tocopherols, it is easy to separate the tocopherols and the tocotrienols, which is industrially very advantageous.

Furthermore, when porous organic polymer particles composed of a (meth) acrylic acid ester-based crosslinked (co) polymer are used as the stationary phase and a hexane-ethanol mixed solvent or the like is used as the mobile phase, tocopherol homologues are obtained. It is possible and preferable to separate α-tocopherol, (β-tocopherol + γ-tocopherol) and δ-tocopherol and also tocotrienols.

The apparatus for carrying out the normal phase liquid chromatography separation performed in the present invention includes a high performance liquid chromatography (HPLC) apparatus, a medium pressure liquid chromatography apparatus, a low pressure liquid chromatography apparatus, etc.
Known devices can be used. Further, in the normal phase liquid chromatography separation carried out in the present invention, a single column method, a multi-stage column method and a plurality of columns are circulated and connected, and a mobile phase flow, sample addition and separation component extraction are provided between the columns. So-called simulated moving bed (simulated moving bed, simulated moving)
Bed or SMB) method and improved simulated moving bed (Im
probed Simulated Moving Be
d or ISMB) method or the like is possible.

[0021]

EXAMPLES Hereinafter, specific embodiments of the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples as long as the gist thereof is not exceeded.

Example 1 Using a high performance liquid chromatography (HPLC) device equipped with a liquid feed pump, an auto sample injector, a column oven, an ultraviolet absorption detector, and a data processing device, a (meth) acrylic acid ester-based cross-link was used as a stationary phase. MCIGEL CHP2MG (manufactured by Mitsubishi Chemical Corporation), which is a (co) polymer, was used as the porous organic polymer particles. A normal phase was used in which a column in which the particles were packed in a stainless steel column having an inner diameter of 4.6 mm and a length of 150 mm and n-hexane / ethanol = 98/2 (volume ratio) having a lower polarity than the stationary phase was used as a mobile phase. Under the liquid chromatography conditions, the liquid was passed through the liquid feed pump at a flow rate of 1.0 ml / min. The temperature of the column oven was 25 ° C.

As a sample, dl-α-tocopherol, d
l-β-tocopherol, dl-γ-tocopherol,
Each 1 mg of dl-δ-tocopherol, dl-α-tocotrienol, dl-β-tocotrienol, dl-γ-tocotrienol, and dl-δ-tocotrienol (above, Calbiochem) was dissolved in 1 ml of n-hexane. 10 μl of each was injected into the column with an auto sample injector. The chromatogram obtained by setting the wavelength of the ultraviolet absorption detector at 295 nm was recorded by the data processor. As a sample for calculating the void volume, 2 μl of carbon tetrachloride dissolved in 1 ml of n-hexane was used, and 20 μl was injected by an auto sample injector, and the wavelength of the ultraviolet absorption detector was 230 nm.
The chromatogram obtained as was recorded with a data processor.

The retention ratio (k ') for each sample was calculated by the following formula.

## EQU1 ## k '= [(elution capacity of each sample)-(elution capacity of carbon tetrachloride)] / [(elution capacity of carbon tetrachloride)] Retention time of each sample, retention ratio of each sample (k' ) Is shown in Table 1. From Table 1, it can be seen that in this example, the retention of the tocotrienols is far from the retention of the tocopherols, so that the tocopherols and the tocotrienols can be easily separated from the solution containing the tocopherols and the tocotrienols.

[0025]

[Table 1]

Example 2 Normal phase chromatographic separation was carried out under the same conditions as in Example 1 except that Orizatocotrienol-30G (manufactured by Oriza Yuka) was used as the sample. Oryzatocotrienol-30G is derived from rice seeds and contains d-α-tocopherol, d-γ-tocopherol and d-α-tocotrienol, d-γ-tocotrienol as main components. As a sample, a solution prepared by dissolving 50 mg of oryzatocotrienol-30G in 1 ml of n-hexane was used, and 5 μl was injected into the column by an auto sample injector. The elution curve of the chromatogram obtained by setting the wavelength of the ultraviolet absorption detector at 295 nm is shown in FIG. Figure 1
Among them, peak 1 represents d-α-tocopherol, peak 2 represents (d-β-tocopherol + d-γ-tocopherol), peak 3 represents d-δ-tocopherol, and peak 4 represents d-tocotrienol. Comparison with the results of Example 1 shows that d-α-tocopherol, (d-β-tocopherol + d-γ-tocopherol) and d-δ-tocopherol are separated, and d-tocotrienols are separated. .

Example 3 HPL equipped with a liquid feed pump, an auto sample injector, a column oven, an ultraviolet absorption detector, and a data processing device
Using a C device, a styrene-divinylbenzene-based crosslinked copolymer pt-butoxystyrene / divinylbenzene = 15/85 (weight ratio) crosslinked copolymer (particle size: 50
˜150 μm, specific surface area (by nitrogen adsorption method): 616
m ² / g, pore volume: 1.22 ml / g, most frequent radius:
Porous organic polymer particles in which t-butoxy groups of 81 angstroms) were converted into hydroxyl groups by alkaline hydrolysis in the usual way were used as stationary phases. A normal phase liquid chromatograph using a column in which these particles are packed in a stainless steel column having an inner diameter of 10 mm and a length of 250 mm and using n-hexane / ethanol = 99/1 (volume ratio) having a lower polarity than the stationary phase as a mobile phase. Under the graphographic conditions, the solution was pumped at a flow rate of 2.4 ml / min. The temperature of the column oven was 25 ° C.

As a sample, dl-α-tocopherol,
dl-β-tocopherol, dl-γ-tocopherol, dl-δ-tocopherol, dl-α-tocotrienol, dl-β-tocotrienol, dl-γ-tocotrienol, dl-δ-tocotrienol (above, Calbiochem) 1 mg each Each of them was dissolved in 1 ml of n-hexane, and 80 μl of each was injected into the column with an auto sample injector. The chromatogram obtained by setting the wavelength of the ultraviolet absorption detector at 295 nm was recorded by the data processor. Further, as the sample for calculating the void volume, 2 μl of carbon tetrachloride dissolved in 1 ml of n-hexane was used, and 100 μl was injected by an auto sample injector, and the wavelength of the ultraviolet absorption detector was 230 nm.
The chromatogram obtained as was recorded with a data processor.

Table 2 shows the retention time of each sample and the retention ratio (k ') of each sample. It can be seen from Table 2 that the retention of tocotrienols is far from the retention of tocopherols in this example as well. In this example, the retention ratio of each sample is smaller than that in Example 1. However, even in such a case, by adopting the simulated moving bed method or the improved simulated moving bed method or the like, it is possible to obtain from the raw material containing the tocotrienols and the tocopherols. Industrially efficient separation of tocopherols and tocotrienols is possible.

[0030]

[Table 2]

Comparative Example 1 Using the same HPLC apparatus as in Example 1 and a column of the same size packed with the same stationary phase, under reversed phase liquid chromatography conditions using isopropanol having a higher polarity than the stationary phase as the mobile phase, Flow rate of 0.5 ml with liquid delivery pump
The solution was passed at a rate of / minute. The temperature of the column oven was 25 ° C. 50 mg oryzatocotrienol-3 as a sample
A solution obtained by dissolving 0G in 1 ml of n-hexane was used, and 10 μl was injected using an auto sample injector. The elution curve of the chromatogram obtained by setting the wavelength of the ultraviolet absorption detector at 295 nm is shown in FIG. In comparison with Example 1, two broad peaks overlap in the chromatogram, indicating that the separation of d-tocopherols and d-tocotrienols is insufficient.

Comparative Example 2 Using the same HPLC apparatus as in Example 1, ULTRASPHERE-S which is a silica gel packed column as a stationary phase.
i (particle diameter 5 μm; column: inner diameter 4.6 mm, length 25)
0 mm, manufactured by ALTEX), and n-hexane / ethanol having a lower polarity than the stationary phase as a mobile phase = 99/1
Under normal phase liquid chromatography conditions using (volume ratio), the solution was pumped at a flow rate of 1.0 ml / min. The temperature of the column oven was 25 ° C. Dl as a sample
-Α-tocopherol, dl-β-tocopherol, d
l-γ-tocopherol, dl-δ-tocopherol,
dl-α-tocotrienol, dl-β-tocotrienol, dl-γ-tocotrienol, and dl-δ-
1m each of tocotrienol (all manufactured by Calbiochem)
Each of 5 g was dissolved in 1 ml of n-hexane, and 5 μl of each was injected with an auto sample injector. The chromatogram obtained by setting the wavelength of the ultraviolet absorption detector at 295 nm was recorded by the data processor.

As the sample for calculating the void volume, 2 μl of carbon tetrachloride dissolved in 1 ml of n-hexane was used, and 10 μl was injected by an auto sample injector, and the wavelength of the ultraviolet absorption detector was obtained as 230 nm. The obtained chromatogram was recorded by the data processor. Table 3 shows the retention time of each sample and the retention ratio (k ') of each sample.
As is clear from Table 3, the retention ratio of each sample is dl-α-
Tocopherol → dl-α-tocotrienol → dl-
β-tocopherol → dl-γ-tocopherol → dl
-Β-tocotrienol → dl-γ-tocotrienol → dl-δ-tocopherol → dl-δ-tocotrienol becomes larger in this order, so in order to separate tocopherols and tocotrienols from a raw material containing tocotrienols and tocopherols, It is necessary to separate each component into small pieces, which is not industrially efficient.

[0034]

[Table 3]

[0035]

In the prior art, it was difficult to industrially separate tocopherols and tocotrienols, but according to the method of the present invention, each of the tocopherols can be produced by an industrially efficient and stable method. Not only the separation of homologues,
It is also extremely useful because it enables separation from tocotrienols.

[Brief description of drawings]

FIG. 1 Oryzatocotrienol-in Example 2
The elution curve by 30 G normal phase chromatography is shown.

FIG. 2 Oryzatocotrienol-in Comparative Example 1
The elution curve by reverse-phase chromatography of 30G is shown.

[Explanation of symbols]

1 peak of d-α-tocopherol 2 peak of d-β-tocopherol + peak of d-γ-tocopherol 3 peak of d-δ-tocopherol 4 peak of d-tocotrienols

Claims (10)

[Claims] 1. A method for separating tocopherols from a liquid containing tocopherols by normal phase liquid chromatography, which comprises using porous organic polymer particles as a stationary phase. 2. A method for separating tocotrienols from a liquid containing tocotrienols by normal phase liquid chromatography, which comprises using porous organic polymer particles as a stationary phase. 3. The tocopherols-containing liquid contains two or more kinds selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol, and α-tocopherol, ( The separation method according to claim 1, wherein β-tocopherol + γ-tocopherol) and δ-tocopherol are separated. 4. A method for separating tocopherols and tocotrienols from a solution containing tocopherols and tocotrienols by normal phase liquid chromatography, characterized in that porous organic polymer particles are used as a stationary phase. . 5. Tocopherols include α-tocopherol, β-tocopherol, γ-tocopherol and δ.
-Tocopherol, comprising two or more species selected from the group consisting of, and α-tocopherol, (β-tocopherol + γ-tocopherol), δ-tocopherol and tocotrienols are separated by normal phase liquid chromatography. 4. The separation method according to 4. 6. The separation method according to claim 1, wherein the porous organic polymer particles are made of a polyvinylbenzene-based crosslinked (co) polymer. 7. The separation method according to claim 1, wherein the porous organic polymer particles are composed of a (meth) acrylic acid ester-based crosslinked (co) polymer. 8. The separation method according to claim 6, wherein the porous organic polymer particles are composed of a hydrolyzate of a pt-butoxystyrene-divinylbenzene cross-linked copolymer. 9. porous organic polymeric particles, the specific surface area (nitrogen adsorption method) of 50m ² / g~2000m ² / g, pore volume (nitrogen adsorption method) of 0.1ml / g~3.0ml / 9. The separation method according to claim 1, wherein g is g. 10. The separation method according to claim 1, wherein hexane or a hexane-ethanol mixed solvent is used as a mobile phase in normal phase liquid chromatography.