JP2000072713A - Separating agent for unsaturated fatty acid and its analog substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a separating agent useful for separating, purifying or analyzing an unsaturated fatty acid and its analog substance, having a high efficiency and high durability. SOLUTION: This separating agent comprises a metal phosphate containing at least silver as a metal component or one or more metal phosphates of silver and one or more metal components composed of metals separated from calcium, zirconium, titanium, yttrium, lantern, aluminum and silicon.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel separating agent used for separating and purifying or analyzing unsaturated fatty acids and their related substances.

[0002]

BACKGROUND ART As a separating agent for unsaturated fatty acids, some separating agents in which silver ions are supported on a carrier such as silica gel have been proposed. The silver ions are carbon-
It is used as a separating agent for unsaturated fatty acids by utilizing the property of exhibiting high affinity for carbon double bonds.
For example, a separating agent in which silver nitrate is impregnated into an inorganic powder such as silica gel, alumina, zeolite or kaolin to physically support silver nitrate (JP-A-63-208549, JP-A-2-8298) or a layered silicate Minerals (clay minerals)
Separating agent in which silver ions are carried by an ion exchange reaction between layers of the same (JP-B-8-13776, JP-A-5-287)
No. 295), a separating agent in which silver ions are supported on a silanol group present on the surface of silica gel by an ion exchange reaction (JP-A-5-317699), and a silver ion is reacted on a sulfone group chemically modified on the silica gel surface. Separating agent supported by ion exchange reaction (EA Emken
et al., J. Am. Oil Chem. Soc., 55 .
(1978) 561). These separating agents are used as column packing materials in liquid chromatography or supercritical chromatography or as a purifying agent in batch processing.

However, in a separating agent in which silver nitrate is physically supported on silica gel or the like (JP-A-63-208549, JP-A-2-8298), the silver nitrate is relatively easily eluted, so that the durability is high. And the degree of separation and the recovery were also problematic. In addition, silver ions are converted to silica gel by an ion exchange reaction (JP-A-5-3176).
No. 99) and layered silicate minerals (Japanese Patent Publication No. 8-1377).
No. 6, JP-A-5-287295), since the ion exchange reaction is a reversible reaction, silver ions are naturally eluted by repeatedly using the separating agent, and accordingly, silver ions are eluted. Adsorption capacity and separation capacity,
Recovery rate etc. decrease. As described above, the conventional separating agent has low durability, and it has been difficult to use the separating agent stably repeatedly.

In recent years, α-linolenic acid, γ-linolenic acid,
Polyunsaturated fatty acids such as dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid have been found to be precursors of physiologically active substances such as prostaglandins, and have been found to be highly unsaturated as pharmaceuticals and health foods. Research and development on the use of saturated fatty acids are being activated. Regarding unsaturated fatty acids with a low degree of unsaturation, the importance of linoleic acid as an essential fatty acid and the fact that erucic acid, which is often contained in rapeseed oil, has adverse effects such as heart damage have been studied for a long time. It is expected that research and development of uses for unsaturated fatty acids having various physiological activities will be further developed. However, it is difficult to obtain unsaturated fatty acids of high purity in large quantities, and this is an obstacle to the progress of basic research, applied research, and practical use. Under these circumstances, development of a new separating agent for separating and analyzing or separating and purifying unsaturated fatty acids and related substances has been desired.

[0005]

DISCLOSURE OF THE INVENTION In view of the above-mentioned background art, the present inventors have intensively developed a separating agent for separating and purifying or analyzing unsaturated fatty acids and their related substances.
It has been found that silver or a metal phosphate comprising silver and some other metal is an excellent separating agent for unsaturated fatty acids. That is, the present invention provides a separating agent for unsaturated fatty acids and related substances characterized by comprising one or more selected from metal phosphates containing at least silver as a metal component, In addition, the above-mentioned metal phosphate is composed of a metal selected from calcium and zirconium, titanium, yttrium, lanthanum, aluminum or silicon as a metal component other than silver and silver, and unsaturated fatty acids and analogs thereof. It provides a separating agent.

Hereinafter, the present invention will be described in detail. Metal phosphate containing at least silver as the metal component according to the present invention
(Hereinafter, abbreviated as silver phosphate) and a separating agent for unsaturated fatty acids and their related substances (hereinafter, referred to as unsaturated fatty acids including these), as compared with conventional separating agents, are shown below. Has advantages. First, the durability of the separating agent is improved, and the separating agent can be repeatedly and stably used for a long period of time. The conventional separation agent in which silver nitrate is supported on silica gel or alumina has the problem that silver ions are easily eluted from the separation agent due to the presence of silver nitrate on the surface of the carrier, and the reproducibility in separation analysis or separation purification is low. was there. Also,
It is said that the separation agent in which silver ions are supported on a carrier such as silica gel by ion exchange reaction has relatively little elution of silver ions.However, since the ion exchange reaction is a reversible reaction, it is considered in principle that Elution of silver ions cannot be avoided.

On the other hand, when the silver phosphate salt of the present invention is used as a separating agent, silver in the salt exists in the form of a physicochemically stable salt. In general, the solubility in a polar organic solvent, a non-polar organic solvent, and water used as a solvent when separating unsaturated fatty acids is very low, so that elution of silver from a silver phosphate salt is extremely small. Therefore, the silver phosphate salt of the present invention has an advantage that the ability to separate and adsorb unsaturated fatty acids is less reduced, and that it can be used stably for a long period of time. Second, the silver phosphate salt of the present invention has an advantage that it can be heat-treated at a high temperature of several hundred degrees or more by utilizing the property of being thermally stable. As a result, the separating agent can be processed into various shapes, such as pellets and filters, depending on the purpose of use.At that time, heat treatment at a high temperature of several hundred degrees or more increases the mechanical strength of the separating agent. It is possible to produce a robust separating agent which is increased and hardly destroyed. A third advantage is that silver phosphate is inactive against low molecular weight organic substances other than unsaturated fatty acids. Note that the low molecular weight organic substance here is an organic substance having a molecular weight of about 10,000 or less.

It is known that silica gel, which is usually used as a carrier in conventional separation agents, strongly adsorbs basic substances and often causes irreversible adsorption. Various organic substances in the sample are adsorbed on the surface of the silica gel, which is used as a carrier of the separation agent in the prior art, where silver nitrate or silver ions are not supported. Class separation is hindered. On the other hand, the silver phosphate of the present invention is inactive against low molecular weight organic substances other than unsaturated fatty acids, so that other components in the sample are not adsorbed. Therefore, when the silver phosphate of the present invention is used as a separating agent, a high separating ability can be achieved with respect to the separation of unsaturated fatty acids.

The separating agent according to the present invention may be used in the form of various known solid carrier materials such as glass, ceramics, metals and resins coated with a silver phosphate salt, or the silver phosphate salt itself may be used. Depending on the purpose of use, any form of use of molding into a desired particle size or shape can be adopted. That is, the separating agent according to the present invention can be roughly classified into the following two embodiments. Separating agent in which the surface of the solid carrier material is coated with silver phosphate salt Separating agent consisting of silver phosphate salt itself

The silver phosphate of the present invention may be a solid (crystalline solid) composed of crystallites grown to such an extent that a diffraction peak is observed in X-ray diffraction, or a clear peak in X-ray diffraction. It may be a solid (amorphous solid) composed of crystallites that are too small to be observed, and the size itself of the crystallite constituting the silver phosphate salt is not particularly limited.

The phosphoric acid composition of the silver phosphate when used for coating the surface of a solid carrier material substance includes orthophosphate,
Any of condensed phosphates such as pyrophosphoric acid, metaphosphoric acid, polyphosphoric acid, and ultraphosphoric acid, and phosphate glass may be used, or a combination of two or more of these may be used. In addition, silver alone may be used as the metal component, and another metal other than silver may be present. By combining silver and a metal element other than silver, the thermal stability of the separating agent can be improved, or the adsorption capacity of unsaturated fatty acids can be adjusted by changing the type and mixing ratio of the metal component other than silver. it can. Metals other than silver used for such purposes include, in particular, magnesium, alkaline earth metals,
Iron group metals, manganese, copper, zinc, zirconium, titanium, yttrium, lanthanum, cerium, aluminum, platinum, palladium, ruthenium, silicon and the like can be used, but in particular calcium, zirconium, titanium, yttrium, lanthanum, Aluminum or silicon is preferred. Note that silicon is not classified as a metal element academically, but since the separating agent according to the present invention has the same function as other metal elements, it is assumed that silicon is included as the metal element in the present invention.

The form of the separating agent can take various forms, such as particles, fibers, pellets, membranes, and filters, depending on the intended use and application. For example, when a separating agent is filled in a chromatographic tube and used as a liquid chromatography column, it is desirable that the separating agent be in the form of particles. The shape of the particles may be spherical or crushed, but for such a purpose, it is preferable to use spherical particles as the separating agent. Generally, when packed and used in a column for high performance liquid chromatography, which is mainly used for analysis, particles having a particle size of about 2 to 20 μm are suitable in order to enhance the resolving power. In pressure chromatography or open column chromatography, particles having a particle size of about 20 to 200 μm are preferable in order to enhance liquid permeability. However, separation agent particles having a particle diameter outside this range can also be used.

Further, such particles can be expected to be used not only as liquid chromatography columns but also as packing materials for supercritical chromatography columns and fluidized bed type chromatography columns. Further, as the separating agent for thin layer chromatography, particles having a size of several μm or more are suitable in order to enhance the coating property of the separating agent on a glass plate or the like. Such a separating agent can be used as a plate for thin layer chromatography by applying it to a glass plate or the like, and is useful as a simple method for separating unsaturated fatty acids. When it is intended to separate and purify unsaturated fatty acids by batch processing, the separating agent may be in the form of pellets in addition to particles. In a batch method in which a separating agent is put into a large amount of sample solution and the target substance is adsorbed on the separating agent and separated and recovered, a stirrer or the like is used to increase the adsorption rate of unsaturated fatty acids dissolved in the liquid to the separating agent. Mechanical agitation is required.

At this time, the separating agent is liable to be destroyed. In this case, the separating agent composed of a pellet-shaped sintered body that has been subjected to a high temperature treatment does not break due to stirring, and has high sedimentation properties, and is easy to separate and recover. This is preferable. When a large amount is to be purified quickly, it is preferable to process the separating agent into a filter and use it as a separating agent in the form of a filter. When the sample solution passes through the filter, unsaturated fatty acids in the sample are collected on the filter. The collected unsaturated fatty acids can be recovered by flowing an eluent through a filter, which is useful as a rapid purification method.

The surface state of the separating agent may be non-porous or porous. However, from the viewpoint of increasing the holding amount of the target substance, the porous material has a higher retention per unit weight of the separating agent. It is efficient because of its large volume. On the other hand, when performing analysis by high performance liquid chromatography, the analysis time can be shortened by using a non-porous separating agent as a column packing material.

Regarding the bond between the silver phosphate salt and the solid carrier material, whether it is a chemical bond or a physical bond, the silver phosphate coated on the surface of the solid carrier material can be used as a separating agent for unsaturated fatty acids. The method and condition of the coating are not particularly limited as long as the function is achieved. For example, the surface of the solid carrier material may be covered with silver phosphate in the form of a film to form a layer having a certain thickness, and the surface of the solid carrier material may be bonded with silver phosphate fine particles spread over the surface. You may. Also, the layer and the binding fine particles can take various forms from a dense and non-porous one to a porous one,
These embodiments can be arbitrarily selected according to the use conditions of the separating agent. For example, if the solid carrier material is physicochemically stable under the conditions for chromatography and does not cause harmful effects with the mobile phase or sample, the surface of the solid carrier material is not necessarily dense with a layer of silver phosphate. It does not need to be covered.

In the case of preparing a separating agent consisting of only a silver phosphate salt, for example, a silver orthophosphate precipitate can be obtained by mixing an aqueous solution of silver nitrate and an aqueous solution of ammonium phosphate. Further, a slurry in which fine particles of silver zirconium phosphate are dispersed can be obtained by mixing an aqueous solution of zirconyl nitrate and silver nitrate with phosphoric acid.
This slurry can be formed into a spherical shape by a known method such as a spray drying method and used as a separating agent suitable as a packing material for chromatography. In addition, the particles mixed with an appropriate organic binder are pressure-formed into a disk shape, and when sufficiently baked at a high temperature, the particles are firmly bonded by sintering,
In addition, it is possible to obtain a disc-shaped sintered body having a large number of pores generated by the organic binder being dispersed. This disc-shaped sintered body is suitable for use as a filter-type separating agent. When producing a separating agent in a form in which the surface of a solid carrier material is coated with a silver phosphate salt,
A method of attaching a silver phosphate salt slurry to the surface of a solid carrier material substance by spraying and heating and drying, or immersing a solid carrier material in a solution or slurry of silver and phosphoric acid or silver and another metal component and phosphoric acid Thereafter, it can be prepared by any of various methods such as a method of separating from a solution or a slurry, drying, and heating to form a layer of the silver phosphate on the surface by heating.

Solvents used for separating and analyzing or separating and purifying unsaturated fatty acids using the separating agent in the present invention include non-polar organic solvents such as n-hexane and cyclohexane, i-propyl alcohol and acetone. And a polar organic solvent such as acetonitrile, water, and the like. Usually, a non-polar organic solvent is used as a solvent for retaining unsaturated fatty acids in the separating agent, and the retained sample is eluted from the separating agent by adding a polar organic solvent thereto to increase the polarity of the solvent.

Since the solubility of the silver phosphate in polar organic solvents, non-polar organic solvents and water is extremely low, various organic solvents can be used in addition to the above-mentioned organic solvents, but they are less harmful to the human body. It is preferable to use n-hexane as the nonpolar solvent and i-propyl alcohol as the polar solvent because of their high solubility and mutual solubility. For example, when a separating agent is used as a packing material for liquid chromatography, a sample containing unsaturated fatty acids is introduced into a column packed with the separating agent, and then n-hexane is passed through to separate saturated fatty acids and the like into the column. The unsaturated fatty acids can be eluted from the column by passing through n-hexane to which several percent to several tens percent of i-propyl alcohol has been added. By appropriately setting the i-propyl alcohol concentration at this time, different unsaturated fatty acids can be separated. The same separation can be performed using a solvent other than n-hexane or i-propyl alcohol.

[0020]

The present invention will be specifically described below with reference to examples and comparative examples of the present invention. However, the present invention is not limited to the following examples.

Example 1 An aqueous silver nitrate solution and phosphoric acid were mixed so that the Ag / P molar ratio was 3/1, and nitric acid was further added to prepare an acidic aqueous solution having a silver orthophosphate composition. Silica gel (for column chromatography,
Spherical, particle diameter: 100-210 μm)
The mixture was stirred gently at room temperature. The silica gel was filtered off, dried and then heat-treated at 450 ° C. for 6 hours. The silica gel particles were analyzed using powder X-ray diffraction and an X-ray microanalyzer to confirm the formation of silver orthophosphate coated on the silica gel. These particles are referred to as silver phosphate-coated silica gel.

A column was prepared by charging 30 g of the silver phosphate-coated silica gel prepared as described above into a glass chromatograph tube having an inner diameter of 2 cm and a length of 30 cm under a hydrostatic pressure under a hydrostatic pressure. A column of a fatty acid ethyl mixture (ethyl esterified crude product from sardine oil, containing 38% of ethyl eicosapentaenoate and 4% of ethyl docosahexaenoate) was introduced into the column, and then n-hexane (1%) was used as an eluent.
00%), n-hexane (95%) / i-propyl alcohol (5%), and n-hexane (80%) / i-propyl alcohol (20%). The fractions collected when the respective eluents were passed through were concentrated, and the components contained in the respective fractions were examined by capillary chromatography. As a result, n-hexane (95%) / i
The fraction eluted with -propyl alcohol (5%) contained 92% pure ethyl eicosapentaenoate, and the fraction eluted with n-hexane (80%) / i-propyl alcohol (20%) contained docosahexaenoic acid. 96% pure ethyl
Was confirmed to be purified. In addition, ethyl eicosapentaenoate and ethyl docosahexaenoate were almost quantitatively recovered, and no nonspecific adsorption phenomenon of unsaturated fatty acids to silver phosphate-coated silica gel was observed.

Comparative Example 1 For comparison, silica gel supporting silver nitrate was prepared by a conventional method, and the purity of ethyl eicosapentaenoate and ethyl docosahexaenoate obtained by the same purification operation as in Example 1 was compared.

The silica gel used in Example 1 (for column chromatography, spherical, particle size: 100 to 210 μm)
m) 30 g of a slurry was charged under hydrostatic pressure into a glass chromatography tube having an inner diameter of 2 cm and a length of 30 cm. 10 in the column
500 ml of an aqueous solution of silver nitrate was passed, and then 500 ml of methanol and 500 ml of n-hexane were passed to prepare a silica gel column supporting silver nitrate. The fatty acid ethyl mixture used in Example 1 (ethyl esterified crude product of sardine oil, containing 38% of ethyl eicosapentaenoate and 4% of ethyl docosahexaenoate) used in Example 1 was introduced into this column, and then the eluent was used. As n-hexane (1
00%), n-hexane (95%) / i-propyl alcohol (5%), and n-hexane (80%) / i-propyl alcohol (20%). In the same manner as in Example 1, as a result of examining the components contained in the fractions collected when the respective eluents were passed, n-hexane (95%
%) / I-propyl alcohol (5%) in the fraction eluted with ethyl eicosapentaenoate having a purity of 65% and n
-Hexane (80%) / i-propyl alcohol (20
%), Ethyl docosahexaenoate was purified with a purity of 30%.

Example 2 Using the column prepared in Example 1, the purification operation of ethyl eicosapentaenoate and ethyl docosapentaenoate shown in Example 1 was repeated to evaluate durability. The column packed with the silica gel coated with silver phosphate was able to be used stably without any decrease in the degree of purification or the recovery rate even in the repeated use test of 50 times.

Comparative Example 2 The same repeated use test as in Example 2 was performed using the silver nitrate-supporting column prepared in Comparative Example 1, and the durability was compared. The column packed with silica gel supporting silver nitrate showed a remarkable decrease in the recovery of ethyl eicosapentaenoate and ethyl docosapentaenoate after repeated use six times.

Example 3 The molar ratio of Ag / P / Zr was 1
Aqueous solution of silver nitrate, aqueous solution of ammonium phosphate, and aqueous solution of zirconyl nitrate were mixed and stirred to obtain a slurry of submicron class fine particles. The slurry was spray-dried to obtain secondary particles having a particle size of several μm to several tens μm. The secondary particles are composed of a collection of submicron class fine particles. From 10 to 2
After classification to 0 μm, heat treatment was performed at 1000 ° C. for 4 hours. The particles were analyzed using powder X-ray diffraction and an X-ray microanalyzer to confirm the formation of silver zirconium phosphate. In Example 3, the particles are referred to as silver zirconium phosphate particles.

Silver zirconium phosphate particles having an inner diameter of 6 mm,
A 250 mm long stainless chromatograph tube was filled with slurry to prepare an HPLC column. A mixture of 20% of methyl eicosapentaenoate (commercially available) and 80% of methyl eicosadienoate (commercially available) was introduced into the column,
As eluents, n-hexane (98%) / i-propyl alcohol (2%) and n-hexane (90%) / i-propyl alcohol (10%) were sequentially passed. The fractions collected when the respective eluents were passed through were concentrated, and the components contained in the respective fractions were examined by capillary chromatography. As a result, n-hexane (98%) /
In the fraction eluted with i-propyl alcohol (2%), only methyl eicosadienate was eluted, and n-hexane (90%) / i-propyl alcohol (10%)
In the fraction eluted with, it was confirmed that methyl eicosapentaenoate was purified to a purity of 98%.

Example 4 The Ag / P / Ti molar ratio was 1
/ 3/2, a methanol solution of silver nitrate, an i-propyl alcohol solution of phosphoric acid (100%), and an i-propyl alcohol solution of titanium isopropoxide are mixed and stirred to form a slurry composed of submicron class fine particles. Obtained. A glass sintered filter was immersed in this slurry for 10 minutes and dried at 80 ° C. for 1 hour. After repeating the operation of immersing the filter in the slurry and drying three times,
Heat treatment was performed at 850 ° C. for 6 hours. The surface layer of the filter was analyzed using powder X-ray diffraction and an X-ray microanalyzer, and it was confirmed that the surface of the glass sintered filter was covered with silver titanium phosphate. This filter is referred to as a silver-titanium phosphate coated filter in Example 4.

A silver phosphate-coated filter was attached to a suction bottle, and a fatty acid methyl mixed sample (mixed at a ratio of 20% of methyl arachidonic acid (commercially available) and 80% of linoleic acid (commercially available)) was attached while suctioning with a water jet pump. n-hexane (99
%) / I-propyl alcohol (1%) was poured into a filter, and the filtrate was collected. Next, n-hexane (70%) / i-propyl alcohol (30%) was similarly poured into the filter, and the filtrate was recovered.
After concentrating each of the filtrates, the components contained in the filtrates were examined by capillary chromatography. as a result,
Methyl arachidonic acid was found only in the fraction recovered with n-hexane (70%) / i-propyl alcohol (30%), confirming that it was purified with a purity of 94%.

Example 5 Silver oxide and diammonium hydrogen phosphate were mixed at a molar ratio of Ag / P = 2/1 and heat-treated at 550 ° C. for 6 hours. The product is ground in an agate mortar and
The particle size was adjusted from 0 to 200 mesh. Product powder X
From the results of the line diffraction, formation of silver pyrophosphate was confirmed. In Example 5, the particles are referred to as silver pyrophosphate particles. The silver pyrophosphate particles prepared as described above were dissolved in a mixed sample of methyl eicosapentaenoate (20%) and methyl eicosadienoate (80%) used in Example 3 and dissolved in n-hexane (98%) / i- It was placed in propyl alcohol (2%) and stirred gently for 1 hour. The silver pyrophosphate particles were separated by filtration, sufficiently washed with n-hexane (98%) / i-propyl alcohol (2%), and then washed with i-propyl alcohol (100%). The fraction collected during washing with i-propyl alcohol (100%) was concentrated and then examined by capillary chromatography. As a result, it was confirmed that ethyl eicosapentaenoate was purified to a purity of 95%. Note that no destruction of silver pyrophosphate particles was observed in a series of steps.

Example 6 An aqueous solution of calcium nitrate and silver nitrate and an aqueous solution of phosphoric acid were subjected to Ca / P / Ag = 10/6.
The resulting slurry mixed at a molar ratio of /0.1 was spray pyrolyzed to obtain spherical particles. The resulting spherical particles were heat-treated at 600 ° C. for 6 hours, and then classified to a size of 4 to 10 μm. The particles were further heat treated at 800 ° C. for 6 hours. As a result of powder X-ray diffraction and X-ray microanalyzer analysis of the particles, formation of apatite containing silver was confirmed. In Example 6, the particles are referred to as Ag-HAP.

The Ag-HAP particles were made to have an inner diameter of 4 mm and a length of 15
Fill a 0mm stainless steel chromatographic tube with slurry,
An LC column was made. A mixture of fatty acid ethyl (20% ethyl docosahexaenoate and 80% ethyl oleate mixed) was introduced into the column, and n-
Hexane (100%), n-hexane (70%) / i-
Propyl alcohol (30%) was passed sequentially. The fractions collected when the respective eluents were passed through were concentrated, and the components contained in the respective fractions were examined by capillary chromatography. As a result, n-hexane (100
%), No ethyl docosahexaenoate was observed in the fraction eluted with n-hexane (70%) / i-propyl alcohol (30%). It was confirmed that it was done.

[0034]

As is apparent from the above description, the unsaturated fatty acid separating agent according to the present invention has various physiological activities. It is a separation agent that has excellent durability and can be used stably for separation and purification.

 ──────────────────────────────────────────────────の Continuing from the front page (72) Inventor Chiya Inoue 1-7-1 Inari, Soka City, Saitama Prefecture Kanto Chemical Co., Ltd. Central Research Laboratory F-term (reference) 4H006 AA02 AD17 BB11 BB14 BS10 4H059 BA26 BB03 BB06 CA21 CA24 EA21 EA40

Claims (2)

[Claims] 1. A separating agent for unsaturated fatty acids and related substances, comprising one or more selected from metal phosphates containing at least silver as a metal component. 2. An unsaturated fatty acid wherein said metal phosphate comprises silver and a metal selected from calcium, zirconium, titanium, yttrium, lanthanum, aluminum or silicon as a metal component other than silver, and an unsaturated fatty acid thereof. Separating agent for related substances.