JPH07126193A - Purification of higher fatty acid, higher alcohol and aromatic compound - Google Patents

Abstract

PURPOSE:To purify higher fatty acids, higher alcohols and aromatic compounds without using an aromatic non-polar phase by using a filler for reversed phase chromatography capable of recognizing the shape and length of a molecule. CONSTITUTION:The purification of the above compounds is carried out by a liquid chromatography using a filler obtained by bonding a polymer of the formula ((n) is 2-200; R1 is Hor methyl; R2 contains (CH2)mCH3; (m) is 3-21; X is functional group such as propyltrimethoxysilyl; Y is residue such as H) through the terminal X group to a silica gel. This process enables the separation and analysis of higher fatty acids, separation and analysis of higher alcohols and separation and analysis of condensed polycyclic hydrocarbons. Since the supported non-polar phase is free from aromaticity, it does not exhibit unfavorable PI-PI interaction with solute molecules. Since the supported non-polar phase is a polymer bonded to silica gel through one terminal, it has flexibility, changes the molecular orientation with temperature and enables the control of the holding time of solute and the shortening of the analysis time by controlling the temperature.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying higher fatty acids, higher alcohols and aromatic compounds using liquid chromatography.

[0002]

2. Description of the Related Art Liquid chromatography has been widely used in the analysis and separation of organic compounds, pharmaceuticals, foods and the like. In particular, reversed-phase partition chromatography has been attracting attention as an excellent method because of its wide range of target compounds and simple separation mechanism. As a conventional filler for purifying higher fatty acids, higher alcohols and aromatic compounds using reversed phase partition chromatography, for example, a filler whose silica gel surface is depolarized with an octyl group or an octadecyl group is known. Has been.

[0003]

However, in the packing material such as the silica gel particles, the separation is carried out only by utilizing the difference in polarity between the non-polar phase and the elution phase introduced on the silica gel. However, when the polarities of the substances to be separated are similar to each other, there is a drawback that good separation cannot be achieved. Also, when the polarity of the substances to be separated is significantly different or low, excessive retention of the filler is observed,
In this case, a special separation operation called gradient elution is required in order to shorten the analysis time, and there is also a problem that the operation becomes complicated.

On the other hand, as a method for depolarizing silica gel, an example in which an aromatic rigid molecule such as a biphenyl group is introduced has been reported. In this case, not only the separation is caused by the difference in polarity but also the molecular It is performed by identifying differences in aromaticity and shape. However, it is generally considered that the aromaticity of the packing material causes tailing in chromatography and is not preferable. For example, as a packing material for liquid chromatography, styrene having a high aromaticity is used.
Divinylbenzene copolymer particles are known, and although they are used as ion exchangers and packing materials for gel permeation chromatography, their use as packing materials for reversed-phase partition chromatography is extremely limited. This is due to the aromaticity of the styrene-divinylbenzene copolymer and π of the solute.
This is because peak tailing based on −π interaction is often a problem.

It is an object of the present invention to provide a packing material for reversed phase partition chromatography which discriminates the shape and length of a molecule without using an aromatic non-polar phase.
This is achieved because the non-polar phase supported on silica gel has a molecular orientation. Another object of the present invention is to use a filler in which the non-polar phase to be supported is a polymer and one end of the polymer is bonded to silica gel for liquid chromatography to obtain higher fatty acids and higher alcohols. To allow the purification of compounds and aromatic compounds.

[0006]

According to the present invention, the general formula (Formula 1)

[Chemical 1] (In the formula, R _{1 represents a} hydrogen atom or a methyl group. R ₂ includes at least (CH ₂ ) mCH ₃ and _{m represents 3} to 21, preferably 8 to 21. n represents 2 to 200, preferably 5
~ 50. By using a packing material in which the polymer represented by (4) is bonded to silica gel through the terminal X, for purification of higher fatty acids, higher alcohols and aromatic compounds.

The present invention will be described in more detail below. The packing material for reverse phase partition chromatography used in the present invention is
The polymer that can be represented by Chemical formula 1 is a filler obtained by binding to silica gel via the terminal X.

R _{1 in} Chemical formula ₁ is a hydrogen atom or a methyl group. R ₂ is a substituent containing an alkyl group having 4 to 22 carbon atoms. When the number of carbon atoms of R ₂ is less than 4, the hydrophobicity is too small, so that the retention capacity as a packing material for reverse phase partition chromatography becomes small, and the separation performance becomes poor, which is not preferable. Further, the larger the carbon number of R ₂ is, the more R
The molecular orientation between the _two becomes high, and as a result, the separation ability also becomes high, but the compound having 23 or more carbon atoms has a problem that the raw material is difficult to obtain and the melting point is too high to obtain a polymer. Poor sex. Examples of R ₂ represented by the general formula (I) include a butyloxy group, a hexyloxy group, an octyloxy group, a decyloxy group, a dodecyloxy group, a tetradecyloxy group, a hexadecyloxy group,
Ester residues of long chain alcohols such as octadecyloxy group and long chain amines such as butylamino group, hexylamino group, octylamino group, decylamino group, dodecylamino group, tetradecylamino group, hexadecylamino group, octadecylamino group Preferable examples thereof include amide residues and the like, and when used, they can be used alone or as a mixture.

X in the chemical formula 1 is a functional group for bonding the polymer represented by the chemical formula 1 to silica gel. For example, a propyltrimethoxysilyl group which directly reacts with the silica gel and bonds thereto is preferable. . However, when the silica gel is previously aminated, carboxylated, or hydroxylated, a substituent containing a carboxyl group, a hydroxyl group, or an amino group can be used for X, and in this case, a substituent such as dicyclohexylcarbodiimide can be used. The polymer represented by Chemical formula 1 can be bonded to silica gel using a condensing agent. Y in Chemical formula 1 may be any residue as long as it does not particularly affect the chromatographic properties, and a hydrogen atom is preferred.

In the present invention, the polymer represented by Chemical formula 1 is most simply the chemical compound represented by Chemical formula ₂ which satisfies the conditions of R ₁ and R 2.
It is obtained by a telomerization method using a terogen represented by Chemical Formula 3 in which the monomer represented by the formula is taxogen.

[0011]

Embedded image CH ₂ ═C (R ₁ ) CO—R ₂

[0012]

Embedded image X- (CH ₂ ) p-Y

Formula (II) satisfying the conditions of R ₁ and R ₂
As the taxogen represented by, butyl acrylate, butyl methacrylate, butyl acrylamide, butyl methacrylamide, hexyl acrylate, hexyl methacrylate, hexyl acrylamide, hexyl methacrylamide, octyl acrylate, octyl methacrylate, octyl acrylamide, octyl methacrylamide, decyl acrylate, Decyl methacrylate, decyl acrylamide, decyl methacrylamide, dodecyl acrylate, dodecyl methacrylate,
Dodecyl acrylamide, dodecyl methacrylamide,
Tetradecyl acrylate, tetradecyl methacrylate, tetradecyl acrylamide, tetradecyl methacrylamide, hexadecyl acrylate, hexadecyl methacrylate, hexadecyl acrylamide, hexadecyl methacrylamide, octadecyl acrylate,
Preferable examples include octadecyl methacrylate, octadecyl acrylamide and octadecyl methacrylamide. As the telogen represented by Chemical formula 3, X is a functional group capable of directly reacting with silica gel and binding thereto, and Y is a functional group having a high chain transfer constant. The alkyl chain length (p) of the spacer connecting X and Y is not particularly limited, but a spacer having p of 2 to 6 can be practically synthesized. As a terogen represented by Chemical Formula 3 satisfying these conditions,
3-Mercaptopropyltrimethoxysilane is preferably mentioned, but for particles obtained by preamination of silica gel, 3-mercaptobutanoic acid, 5-mercaptohexanoic acid, etc., and particles obtained by pre-carboxylation or hydroxylation of silica gel are used. In contrast, 3-mercaptopropylamine, 6-mercaptohexylamine and the like can be mentioned.

In the present invention, the polymerization degree of the polymer represented by Chemical Formula 1 must be at least 2 to 200. A polymerization degree of 5 to 50 is suitable, and a polymerization degree of 5 to 5 is preferable.
In the polymer of 0, the side chain R ₂ is well molecularly oriented, and as a result, good separability is obtained. A polymer having a degree of polymerization of 51 to 200 also has a good molecular orientation, but the ratio of the residue X in the chemical formula 1 in one molecule decreases, and the reactivity with silica gel deteriorates. In the case of a polymer having a degree of polymerization of 201 or more, not only the proportion of the residue X is remarkably reduced but also the solubility is reduced, so that it is practically impossible to bond to silica gel. The degree of polymerization can be easily adjusted by adjusting the mixing molar ratio of terogen and taxogen when a terogen having a mercapto group having a high chain transfer constant such as 3-mercaptopropyltrimethoxysilane is used.

In the present invention, there are roughly two methods for supporting the polymer represented by Chemical formula 1 on silica gel particles. When X of the polymer represented by the chemical formula 1 can directly react with the silanol residue of silica gel like a trimethoxysilyl group, the polymer supported on silica gel is mixed in an organic solvent and gently stirred. While at 60-80 ℃ 1
The polymer can be bonded to silica gel by keeping it for about 2 hours. As the organic solvent, carbon tetrachloride,
Chloroform, tetrahydrofuran, dioxane and the like can be used. On the other hand, when the polymer X represented by Chemical formula 1 contains a carboxyl group, partially aminated silica gel is used, and when the polymer X represented by Chemical formula 1 contains an amino group or a hydroxyl group, The partially carboxylated silica gel can be used to attach the polymer to the silica gel by conventional condensation methods. As the condensing agent, dicyclohexylcarbodiimide, diethyl cyanophosphate or the like can be used.

In the packing material for reversed phase partition chromatography of the present invention, in order to improve the separation performance, the silica gel serving as a carrier is preferably porous spherical particles. in this case,
The sphere diameter strongly depends on the purpose of separation, but in the case of use mainly for analysis, a sphere having a diameter of several μm to 10 μm and a narrow particle size distribution is used. On the other hand, in the case of use mainly for high-speed large-volume preparative collection, particles having a larger particle size, for example,
Those of 0 μm are used.

The packing material for reversed phase partition chromatography of the present invention can be packed into a column in the same manner as the usual silica gel packing material for reversed phase partition chromatography, such as higher fatty acids, higher alcohols and aromatic compounds. Can be used for purification. Purifiable higher fatty acids,
The higher alcohols and aromatic compounds (condensed polycyclic hydrocarbons) are long chains (which may be linear, branched, or unsaturated) or aromatic rings having 8 to 24 carbon atoms. Any material that can be dissolved in the eluent may be used. In addition, even higher fatty acids, higher alcohols and aromatic compounds having a long chain (which may be any of straight chain type, branched chain type and unsaturated type) or an aromatic ring having 25 or more carbon atoms, which can be dissolved in the eluent It can be purified. Eluents include methanol, distilled water, ethanol, acetonitrile, acetone, acetic acid, carbon tetrachloride,
Chloroform, cyclohexane, 1,2-dichloroethane, dichloromethane, 1,4-dioxane, ethyl acetate, n-heptane, n-hexane, 2-propanol,
Tetrahydrofuran, 2,2,4-trimethylpentane (isooctane) and the like can be preferably mentioned, and they can be used alone or as a mixture when used.

[0018]

EXAMPLES The present invention will be described in more detail with reference to Examples, Test Examples and Comparative Examples, but the present invention is not limited to these.

Example 1 A two-necked Erlenmeyer flask was equipped with a reflux condenser and a nitrogen introducing tube. Octadecyl acrylate (Taxogen)
And 3-mercaptopropyltrimethoxysilane (telogen) were added together with ethanol, and the mixture was stirred for 10 minutes while introducing nitrogen gas. Next, azobisisobutyronitrile was added as an initiator, and the mixture was stirred under a nitrogen gas atmosphere at 80
Radical telomerization was carried out at the charging ratio of telogen and taxogen shown in Table 1 under conditions of 6 ° C. and 6 hours. Allow to cool to room temperature, filter the precipitate on a G-5 glass filter, and wash thoroughly with methanol and acetone.
It was dried under reduced pressure to obtain the desired polymer. The same polymer is hereinafter abbreviated as ODA _n . In this abbreviation, n represents the average degree of polymerization.

[0020]

[Table 1]

The obtained ODA _n was dissolved in deuterated chloroform and the average degree of polymerization n was determined by ¹ H-NMR. In addition, the phase transition temperature of ODA _n was determined by differential scanning calorimetry. Obtained ODA _n and silica gel (dry state)
Were placed in a three-neck round bottom flask with carbon tetrachloride in the mixing ratios shown in Table 2. This was equipped with a stirring seal and a reflux condenser, put in an oil bath at 80 ° C., and stirred for 12 hours. After the completion of stirring, the silica gel was recovered with a G5 glass filter, washed with carbon tetrachloride, and then collected by filtration to obtain silica gel carrying ODA _n . The supported amount was determined by elemental analysis, and the structure of the supported substance was confirmed by infrared absorption spectrum and differential scanning calorimetry analysis.

[0022]

[Table 2]

Examples 2 to 6 The desired polymer was obtained in the same manner as in Example 1, except that the type of taxogen, the charge ratio thereof, the solvent, the reaction temperature, etc. were changed as shown in Table 1. The abbreviations for the polymers were prepared according to the type of taxogen used and are shown in Table 1.
In this abbreviation, n represents the average degree of polymerization. The loading of the polymer on silica gel was carried out in the same manner as in Example 1 by loading the polymer on silica gel.

Example 7 The desired polymer was obtained in the same manner as in Example 1 except that the types of telogen, the charging ratio thereof, the solvent, the reaction temperature and the like were changed as shown in Table 3. The abbreviations for the polymers were prepared according to the type of telogen used and are shown in Table 3. still,
In this abbreviation, n represents the average degree of polymerization.

[0025]

[Table 3]

The obtained polymer and partially aminated silica gel (dry state) were put in a three-round bottom flask with tetrahydrofuran at a mixing ratio shown in Table 2. Dicyclohexylcarbodiimide was added thereto, and a stirring seal and a reflux condenser were attached, and the mixture was stirred at room temperature for 24 hours. After stirring, G5
After collecting with a glass filter and washing with tetrahydrofuran, silica gel carrying a polymer was obtained by filtration. The supported amount was determined by elemental analysis, and the structure of the supported substance was confirmed by infrared absorption spectrum and differential scanning calorimetry analysis.

Examples 8 to 10 The desired polymer was obtained in the same manner as in Example 1 except that the types of telogen, the charging ratio thereof, the solvent, the reaction temperature and the like were changed as shown in Table 3. The abbreviations for the polymers were prepared according to the type of telogen used and are shown in Table 3. still,
In this abbreviation, n represents the average degree of polymerization. Loading of the polymer on silica gel was changed except that the mixing ratio of the polymer and silica gel, the reaction solvent, the temperature, etc. were changed as shown in Table 2.
The polymer was supported on silica gel in the same manner as in Example 7.

Test Example 1 Particle D 3.3 g (dry state), 1-hexanol 15 m
1 and 15 ml of chloroform were placed in a 100 ml beaker and dispersed using an ultrasonic cleaner for about 15 minutes. This was placed in a packer connected to a stainless steel column having a diameter of 4.6 mm and a length of 30 cm, and the void was filled with a mixed solvent of 1-hexanol and chloroform (1: 1), and then the flow rate was 8
Chloroform was flushed at ml / min. Pressure is 450k
The flow rate was adjusted to g / cm ² , and chloroform was further flowed at the same pressure for 30 minutes. Remove the column from the packer, attach a filter, and 100 m at a flow rate of 1 ml / min.
The filling was completed by flushing with 1 l of chloroform and 100 ml of methanol. Liquid chromatography was performed using benzene, butylbenzene, hexylbenzene, and octylbenzene as samples, and methanol / water (9: 1) as an eluent. As shown in FIG. 1, a chromatogram in which all samples were completely separated was obtained.

Test Example 2 After packing the particles with A in the same manner as in Test Example 1,
Liquid chromatography was performed using caprylic acid, lauric acid, palmitic acid, arachidic acid, and lignoceric acid, which are higher fatty acids as samples, and methanol as an eluent. As shown in FIG. 2, it was confirmed that there was no unfavorable peak tailing and that all samples could be completely separated.

Test Example 3 With respect to the column prepared in Test Example 2, the higher alcohols 1-octanol, 1-dodecanol, 1-hexanol, 1-eicosanol, and I-tetracosanol were used as samples, and methanol was used as an eluent. Liquid chromatography was performed using. As shown in FIG. 3, a chromatogram in which all samples were completely separated was obtained.

Test Example 4 The column prepared in Test Example 2 was subjected to liquid chromatography using condensed polycyclic hydrocarbons such as benzene, naphthalene, anthracene, naphthacene and pentacene as samples and methanol as an eluent. . Figure 4
As shown in, a chromatogram in which all samples were completely separated was obtained.

Test Example 5 With respect to the column prepared in Test Example 2, caprylic acid, lauric acid, palmitic acid, arachidic acid and lignoceric acid were used as samples, and methanol / 0.05N was used as an eluent.
Liquid chromatography was performed using acetic acid (19: 1) at different temperatures. As a result, a graph (FIG. 5) showing a special temperature dependence of the retention coefficient (the higher the retention coefficient, the stronger the retaining force) was obtained. That is, it was confirmed that at a temperature at which the non-polar phase supported on silica gel is in a crystalline state (in the case of the particle A, the phase transition temperature is about 38 ° C.), this column has a strong holding power for higher fatty acids. .

Test Example 6 For the column prepared in Test Example 2, 1
Liquid chromatography was carried out using -octanol, 1-dodecanol, 1-hexadecanol, 1-eicosanol, 1-tetracosanol and methanol / water (19: 1) as an eluent at different temperatures. As a result, a graph (FIG. 6) showing a special temperature dependency with respect to the retention coefficient (the higher the retention coefficient, the stronger the retaining force) was obtained. That is, it was confirmed that at a temperature at which the non-polar phase supported on silica gel is in a crystalline state (in the case of particles A, the phase transition temperature is about 38 ° C.), this column has a strong retention force for higher alcohols.

Test Example 7 With respect to the column prepared in Test Example 2, liquid chromatography was carried out by using benzene, naphthalene, anthracene, naphthacene and pentacene as samples and changing the temperature by using methanol as an eluent. As a result, a graph (FIG. 7) showing a special temperature dependency with respect to the retention coefficient (the higher the retention coefficient, the stronger the retaining force) was obtained. That is, at a temperature at which the non-polar phase supported on silica gel is in a crystalline state (in the case of particle A, the phase transition temperature is about 38 ° C.), this column has a strong holding power for condensed polycyclic hydrocarbons. confirmed.

Comparative Example 1 OD as a packing material for commercially available reversed phase partition chromatography
The same examination as in Test Example 5 was performed using S. No significant temperature dependence on the retention factor was confirmed (Fig. 8).
In addition, the ability to retain higher fatty acids was also low.

Comparative Example 2 OD as a packing material for commercially available reverse phase partition chromatography
The same examination as in Test Example 6 was performed using S. No significant temperature dependence on retention factor was confirmed (Fig. 9).
In addition, the ability to retain higher alcohols was also low.

Comparative Example 3 OD as a packing material for commercially available reverse phase partition chromatography
Using S, the same examination as in Test Example 7 was performed. No significant temperature dependence on the retention factor was confirmed (Fig. 1
0). In addition, the retention of condensed polycyclic hydrocarbons was low.

[0038]

INDUSTRIAL APPLICABILITY The packing material for reversed phase partition chromatography of the present invention has a non-polar phase having no aromaticity and having a high molecular orientation, which is carried on silica gel. Reversed-phase partition chromatography can be provided that achieves a separation similar to that of a bulking agent and that utilizes the molecular orientation of the non-polar phase to distinguish the shape and length of the molecule. Specifically, it enables separation and analysis of higher fatty acids, separation and analysis of higher alcohols, and separation and analysis of fused polycyclic hydrocarbons. Furthermore, since the supported non-polar phase has no aromaticity,
It does not show undesired π-π interactions with solute molecules.
Further, the supported non-polar phase is a polymer, and since one end of the polymer is bonded to silica gel, it has flexibility, and therefore exhibits a change in the degree of molecular orientation with temperature.
By utilizing this change in the degree of molecular orientation, it is possible to control the retention time of the solute, and therefore, even in the case of solutes that exhibit excessive retention, by adjusting the temperature without using a special gradient elution method. The analysis time can be shortened.

[Brief description of drawings]

1 shows a chromatogram in Test Example 1 in which the separation of a mixture of benzene, butylbenzene, hexylbenzene and octylbenzene was tested using Particle D.

FIG. 2 shows a chromatogram in Test Example 2 in which the separation of a mixture of caprylic acid, lauric acid, palmitic acid, arachidic acid and lignoceric acid was tested using particles A.

FIG. 3 uses particles A, 1-octanol, 1-dodecanol, 1-hexadecanol, 1-eicosanol,
3 shows a chromatogram in Test Example 3 in which the separation of a mixture of 1-tetracosanol was tested.

FIG. 4 shows a chromatogram in Test Example 4 in which the separation of a mixture of benzene, naphthalene, anthracene, naphthacene and pentacene was tested using particle A.

FIG. 5 shows the results of plotting the retention capacity of particles A for caprylic acid, lauric acid, palmitic acid, arachidic acid, and lignoceric acid against temperature in Test Example 5.

FIG. 6 shows the results of plotting the retention ability of particle A for 1-octanol, 1-dodecanol, 1-hexadecanol, 1-eicosanol, and 1-tetracosanol against temperature in Test Example 6.

FIG. 7 shows the results of plotting the retention ability of particles A for benzene, naphthalene, anthracene, naphthacene, and pentacene against temperature in Test Example 7.

FIG. 8 shows the results of plotting the retention capacity of ODS for caprylic acid, lauric acid, palmitic acid, arachidic acid, and lignoceric acid in Comparative Example 1 against temperature.

FIG. 9 shows the results of plotting the retention capacities of ODS for 1-octanol, 1-dodecanol, 1-hexadecanol, 1-eicosanol, and 1-tetracosanol against temperature in Comparative Example 2.

FIG. 10 shows the results of plotting the retention capacity of ODS for benzene, naphthalene, anthracene, naphthacene, and pentacene against temperature in Comparative Example 3.

[Explanation of symbols]

11: benzene 42: naphthalene 12: butylbenzene 43: anthracene 13: hexylbenzene 44: naphthacene 14: octylbenzene 45: pentacene 21: caprylic acid 22: lauric acid 23: palmitic acid 24: arachidic acid 25: lignoceric acid 31: 1 Octanol 32: 1-dodecanol 33: 1-hexadecanol 34: 1-eicosanol 35: 1-tetracosanol

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07C 233/09 7106-4H C11C 1/08 2115-4H G01N 30/48 K 8310-2J 30/88 C 8310-2J // C07C 31/125 9159-4H

Claims (5)

[Claims] 1. A method for purifying higher fatty acids, higher alcohols, and aromatic compounds using liquid chromatography. 2. The method according to claim 1, wherein the silica gel particles in which the polymer represented by the general formula (Formula 1) is bonded via the terminal X are used as a packing material for liquid chromatography. [Chemical 1] 3. The particle according to claim 2, wherein n in Chemical formula 1 is 2 to 200. 4. The particle according to claim 2, wherein R ₂ in Chemical formula 1 contains at least a (CH ₂ ) _m CH ₃ group in the chemical structure, and m is ₃ to 21. 5. The particle according to claim 2, wherein R _{1 in} Chemical formula ₁ is a hydrogen atom or a methyl group.