METHOD OF CONCENTRATION AND SEPARATION OF UNSATURATED FATTY ACID ESTERS

Abstract: This invention provides a method of concen­ trating and separating γ-linolenic acid esters and/or arachidonic acid esters characterized in that a fatty acid ester mixture containing γ-linolenic acid ester and/or arachidonic acid ester is brought in contact with crystalline aluminosilicate zeolites to make adsorption and desorption. According to this invention, high purity γ-linolenic acid and/or arachidonic acid ester(s) may be obtained on industrial scale.

Description: BACKGROUND OF THE INVENTION Field of the invention The present invention relates to a method of concentration and separation of γ-linolenic acid esters or arachidonic acid esters by taking advantage of the difference in affinity of highly unsaturated fatty acid esters to zeolites. Description of the prior art γ-Linolenic acid (hereinafter abbreviated to GLA) and arachidonic acid (hereinafter abbreviated to AA) are important materials as precursory materials to prostaglandin and are recently drawing particular attention because of their divergent physiological activities. Particularly, as a result of discovery that GLA or AA can be mass-produced utilizing various species of microorganisms, active research and development have been pursued on them. Heretofore known as general methods for concentration and separation of GLA and AA from mixtures of fatty acid esters are urea adduct process and silver complex process (both covered by British Patent No. 1240513); by the former method, the products' purities are low and the urea's cost is high and the latter method also involves high cost of silver; therefore, they both can hardly be said suitable for application on industrial scale. As other potentially effective methods, enzyme method, low temperature fractional crystallization method, supercritical fluid extraction method, molecular distillation method, etc., may be mentioned. Further methods for separation of alkyl fatty acid esters are known from EP-A-0 062 113, EP-A-0 002 545 and EP-A-0 001 854. EP-A-0 062 113 discloses the fractionation of alkyl carboxylate mixtures utilizing selected surface aluminated silica gel adsorbent and selected solvents. A continuous, countercurrent flow, fluid-solid contacting process for separating esters of fatty acids by degree of unsaturation is described in EP-A-0 002 545. EP-A-0 001 854 discloses a process for separating mixtures of fatty acid esters by contacting said mixtures with an X or Y zeolite, thereby selectively absorbing the unsaturated esters onto the zeolite. With these methods, many defects and technical themes to resolve in the aspects of purity, cost and operation are left and therefore, it is quite difficult to obtain GLA and AA at high purity on industrial scale. SUMMARY OF THE INVENTION The object of this invention is to provide a method of concentration and separation of unsaturated fatty acid esters for obtaining GLA and/or AA at high purity on industrial scale. Other objects and advantages of this invention will become apparent from the following detailed description of this invention. The present inventors, as a result of their assiduous study in an effort to attain the aforementioned objective, have found out the fact that the desired high purity GLA esters and AA esters can be readily obtained by making adsorption - desorption operation, using crystalline aluminosilicate zeolites, which finding has led to completion of this invention. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of concentrating and separating γ - linolenic acid esters and/or arachidonic acid esters in which their adsorption and desorption are performed by bringing in contact with crystalline aluminosilicate zeolites a fatty acid ester mixture containing γ -linolenic acid esters and/or arachidonic acid esters, characterized in that said crystalline aluminosilicate zeolite has a molar ratio of SiO₂/Al₂O₃ ranging from 3 to 5.3 and that said desorption of γ -linolenic acid esters and/or arachidonic acid esters is performed by treating with a mixture of a polar solvent and a non-polar solvent in proportion of 0.01 - 10 % by volume of the polar solvent to the nonpolar solvent by column operation. The concentration and separation method of this invention comprises more particularly 1 ○ a process of dissolving in a nonpolar solvent a fatty acid ester mixture containing GLA ester or AA ester and bringing this solution in contact with crystalline aluminosilicate zeolites, thereby selectively adsorbing the GLA ester and the AA ester, then 2 ○ desorbing the GLA ester and the AA ester by contacting the aforementioned crystalline aluminosilicate zeolites with a polar solvent and finally, 3 ○ a process of removing the polar solvent from the GLA ester or AA ester containing solution. The fatty acid esters used according to this invention include esters obtained from lipid containing GLA or AA or both and such lower alcohols as methanol, ethanol, propanol, butanol, and in addition, fatty acid esters of such polyvalent alcohols as glycol, glycerine, Lipid containing GLA and AA may be produced, for example, by such genera of molds as Aspergillus, Fusarium, Penicillium, Mucor, Rhizopus, Mortierella, Cunninghamella, Choanephora, or such genera of algae as Spirulina, Porphyridium, seeds of evening primrose or fish oils, Such lipid is turned into fatty acid esters by undergoing ester exchange with one of the aforementioned alcohols in the presence of an appropriate catalyzer. As the crystalline aluminosilicate zeolites, those with mol ratio of SiO₂/Al₂O₃ ranging from 3 to 5,3 are used according to the present invention from the standpoint of selectivity and rate of adsorption of GLA esters and AA esters. As zeolites which meet these conditions, those available on the market such as Y-type zeolites, mordenite, erionite, ferrielite, L-type zeolites, are usable, but use of crystalline aluminosilicate zeolites which are subjected to such treatments as ion exchange, will result in great improvement in the ability of concentration and separation, depending on the fatty acid composition (or the type of alcohol which is combined with fatty acids as esters) in the fatty acid ester mixture. As the cations which improve the ability of concentration and separation through ion exchange, for example, K⁺, Na⁺, Ag⁺, Cs⁺, Rb⁺, Li⁺, Mn²⁺, Cu²⁺, Ca²⁺, Mg²⁺, Ba²⁺, Be²⁺, Sr²⁺, Fe³⁺, La³⁺, Ce³⁺, Sc³⁺, Y³⁺, NH₄⁺, CH₃NH₃⁺, (CH₃)₂NH₂⁺, C₂H₅NH₃⁺, may be mentioned. These cations are of course effective not only singly but in the state of two or more members coexisting. In this instance, replacing sodium with any of the aforementioned cations until it is reduced to 5.0% by weight or less in the crystal thus produced should be preferable from the standpoint of selectivity and rate of adsorption. Particularly, when separating GLA esters from a mixture of fatty acid ethyl esters or methyl esters, use of Y-type zeolite being sodium Y-type zeolite available on the market which has been subjected to ion exchange with cation(s) including K⁺, Rb⁺, Cs⁺, NH₄⁺, CH₃NH₃⁺, (CH₃)₂NH₂⁺ and C₂H₅NH₃⁺ is very effective from the standpoint of selectivity of GLA esters. The ion exchange of zeolite available on the market may be readily made by a normally well-known method; thus, the specified zeolite is dipped in an aqueous solution of salts containing the cation(s) with which the exchange is desired to be made, e.g., K⁺, Rb⁺, Cs⁺, Ag⁺, Cu²⁺, Mn²⁺, Mg²⁺, Ba²⁺, Fe³⁺, NH₄⁺ or CH₃NH₃⁺., and this solution is held at 70 - 100°C and stirred for about 24 hr; this operation is repeated several times; after this treatment, the zeolites are dried in atomosphere at 120- 700°C or calcined; in this way ion exchanged zeolites are readily obtained. However, heat treatment at high temperatures of NH₄⁺, CH₃NH₃⁺, (CH₃)₂NH₂⁺ and C₂H₅NH₃⁺ involves the possibility of removal of ammonia or inducement of chemical reactions; therefore, the drying temperature range should preferably be on the order of 120°C - 250°C to have any of these cations contained in zeolites. The zeolites used may have whatever form, powders, pellets, beads, granules, etc. As the nonpolar solvents used according to this invention, straight chain or cyclic nonpolar solvents such as n-heptane, n-hexane, cyclohexane, benzene, p-xylene, n-octane, may be utilized; of course, mixed solvents of two or more members of them are usable. As the polar solvents for desorption, wide range of polar solvents such as acetone, methanol, ethanol, chloroform, diethyl ether, ethyl acetate, may be used. And two or more members of these polar solvents in mixture or those prepared by mixing polar solvents and nonpolar solvents to alter the polarity of the polar solvents may be employed. When obtaining high purity GLA esters or AA esters by the later-described column operation method, as the polar solvent used in desorbing fatty acid esters, the aforementioned polar solvents are used mixed with nonpolar solvents at 0.01% - 10% by volume in proportion thereto in regard to selectivity of GLA esters or AA esters. When, for example, n-hexane containing 0.1% ethanol is used as the polar solvent for desorption, GLA esters with purities 90% or higher may be readily obtained. The temperature when fatty acid esters and various solvents are brought in contact with zeolites should normally be on the order of the temperature range (10°C - 90°C) which is used for adsorption operation of solvents. If the temperature is too low, impurities other than GLA esters and AA esters will be adsorbed in large amounts; this is undesirable. On the other hand, at too high temperatures, unsaturated fatty acids tend to be denatured and if the temperatures are higher than the boiling point of the solvent used, the operation need to be performed under pressure, to economic disadvantage. Particularly desirable operation temperature range should be 20°C - 50°C. In the adsorption process of this invention, a fatty acid ester mixture containing GLA ester and AA ester is dissolved in a nonpolar solvent which is 30 times or less in weight ratio thereto. 1 ○ This solution is put in a stirring tank; then, with crystalline aluminosilicate zeolites added 0.05 - 5 times said fatty acid ester mixture in weight ratio thereto, the solution is stirred for 0.5 - 10 hr, thereby making selective adsorption of GLA ester and AA ester. 2 ○ Said fatty acid ester mixture solution is passed through zeolites prefilled in a column in contact therewith, thereby making selective adsorption of GLA ester and AA ester. These methods are applicable. With regard to the amount of the solution passed in the column operation of 2 ○ , normally, the operation may be run with the liquid hourly space velocity (LHSV) in the range of 0.5 - 10, similarly as in the operation with silica gel or ion exchange resin. Or by setting the column operation pressure above atmospheric pressure, e.g., from the intermediate pressures of 10 - 100 kg/cm² to high pressures, this column may be utilized as a compacter separation column. Particularly, when high purity GLA esters or AA esters are desired to have, the column operation is preferred. Then in the desorption process, when the aforementioned 1 ○ is applied, after filtering and fractionating the nonpolar solvent inside the tank, a polar solvent is added 0.5 - 30 times the crystalline aluminosilicate zeolites in weight ratio thereto and the mixed solution is stirred for 0.5 -5 hr; by repeating this treatment about 2 - 5 times, GLA ester and AA ester may be concentrated and separated. And when the aforementioned 2 ○ is applied, GLA ester and AA ester may be desorbed by passing a polar solvent through the crystalline aluminosilicate zeolites column under the condition of a liquid hourly space velocity (LHSV) being 0.05 - 10. In this instance, a polar solvent 10 - 100 times the crystalline aluminosilicate zeolites in weight ratio thereto is passed and the solution flowing out from the column is divided into 2 - 100 fractions, whereby the fatty acid esters may be successively desorbed in the order of increasing affinity to the zeolites, to be separated simultaneously. And if the polar solvent used is not merely one type, but those of various types are used and those with gradually increasing polarities are passed, various types of fatty acid esters may be divided into fraction at high efficiency. Finally, the polar solvent is removed by distilling the fraction with high concentration of GLA ester or AA ester in the divided solution, whereby it is possible to obtain high purity GLA ester and AA ester. Fractions of other fatty acid esters may be similarly recovered. The crystalline aluminosilicate zeolites, after having been fractionated, are washed with a nonpolar solvent; then, such alas as nitrogen is passed therethrough, followed by drying at 100 - 500°C, and so forth. This operation enables its repetitive use. The GLA and AA obtained by the aforementioned operation have very high purity; the separation method is highly advantageous in cost, as compared with conventional methods, and promises enormous contribution in planning its industrialization. In the following, this invention is described particularly in connection with its preferred embodiments. In these examples, percentage (%) means % by weight, unless otherwise specified. Comparative Example 1 A 1N aqueous solution of each of various salts was put in a flask in proportion of 100ml to 5g of sodium Y type zeolite (trade name SK-40) with SiO₂/Al₂O₃ mol ratio approx. 5 manufactured by Union Carbide Corp.; the system was stirred at temperatures of 90 - 100°C for 24 hr, thereby exchanging sodium ions with the other ion(s), followed by water rinsing, and then, the product was dried at 160°C for 1 hr. In this instance, the sodium content in zeolites was set always 5.0% by weight or less. On the other hand, Mucor ambiguus IFO 6472 was subjected to aerobic cultivation at 30°C for approx. 70 hr in a culture of 4% glucose, 0.3% KH₂PO₄, 0.2% (NH₄)₂SO₄, 0.1% (NH₂)₂CO, 0.05% MgSO₄·7H₂O, 0.001% FeSO₄·7H₂O, 0.001% CaCl·2H₂O, 0.00002% CuSO₄·5H₂O, 0.0001% ZnSO₄·7H₂O, 0.0001% MnCl₂·4H₂O, 0.01% NaCl, 0.04% yeast essence, 0.04% malt essence and 0.02% polypeptone, and then, the lipid accumulated in this fungus was extracted by homogenization. The lipid obtained in this way was subjected to ester exchange in 0.5N NaOH methanol solution, yielding a methyl ester mixture of the fatty acid composition shown in Table 1. In Table 1, C n:k represents fatty acid esters defined by the molecular formula C n-1 H 2(n-k)-1 COOR (R : alkyl group). The same applies hereinafter. Five grams of this fatty acid ester mixture was dissolved by addition of 30g of n-heptane; to this solution 2.5g of each of various Y type zeolites prepared as hereabove-described was added and this system was stirred at 25°C for 1 hr, followed by filtration. Thereafter, 30g of chloroform was added to this zeolite, the system was then, stirred at 25°C for 2 hr, followed by filtration, and the filtrate was condensed. The amounts (g) of the total fatty acid esters thus obtained and the fatty acid compositions are shown in Table 2. Jointly listed in Table 2 are results of experiments conducted at the same time by the similar method as hereabove-described using sodium Y type zeolite (trade name SK-40) available on the market, hydrogen Y type zeolite with SiO₂/Al₂O₃ mol ratio approx. 5.5 (trade name TSZ-303PSH2, manufactured by Toyo Soda Co.), sodium mordenite with SiO₂/Al₂O₃ mol ratio ratio approx. 10 (trade name Zeolon 900Na, manufactured by Norton Corp.), sodium X type zeolite with SiO₂/Al₂O₃ mol ratio approx. 2.5 (trade name Molecular Sieves 13X, manufactured by Union Carbide Corp.) Table 2 implies that use of zeolite with SiO₂/Al₂O₃ mol ratio 3 or higher will greatly improve the purity of the GLA esters. Comparative Example 2 Various Y type zeolites were prepared through ion exchange with various cations by the similar method as in Comparative Example 1 using sodium Y type zeolite with SiO₂/Al₂O₃ mol ratio 5.3 (trade name TSZ-320 NAD) manufactured by Toyo Soda Company. The same fatty acid ester mixture as used in Comparative Example 1 was concentrated by the same operation of adsorption -desorption as in Comparative Example 1, using various Y type zeolites. The results are shown in Table 3. In this table , the "ion exchange rate" represents the percentage of Na⁺ contained in the zeolite available on the market which has been exchanged by any of other cations. Comparative Example 3 Five grams of fatty acid methyl ester mixture, the same material as, of Comparative Example 1, was dissolved in 6g of n-heptane and this solution was passed through a glass column 2.1cm in ID and 50cm long filled with 50g of potassium Y type zeolite prepared similarly as in Comparative Example 1. The polarity of the elution solvent was raised little by little from that of 100% n-heptane by successively mixing increasing amounts of chloroform. The column was held at a temperature of 40°C under normal pressure and the elution was conducted at a liquid hourly space velocity of elution solvent of 1.0 (1/hr). The eluate was divided into 10mℓ fractions. The types and amounts of the elution solvents used are given hereunder.: 1 Fraction Nos. 1 - 30 n-Heptane 300mℓ 2 Fraction Nos. 31 - 50 n-Heptane : Chloroform = 4 : 1 200mℓ 3 Fraction Nos. 51 - 70 n-Heptane : Chloroform = 5 : 5 200mℓ 4 Fraction Nos. 71 - 100 n-Heptane : Chloroform = 1 : 3 300mℓ By removing by way of distillation the elution solvent of Fraction Nos. 60 - 64 obtained in this way, 0.20g of fatty acid methyl ester was obtained. The fatty acid composition of the fatty acid esters is shown in Table 4. It indicates that the purity of γ-linolenic acid ester is remarkably upgraded. Example 1 Five grams of methyl ester mixture of the same fatty acid composition as of Comparative Example 1 was dissolved in 6g of n-hexane and this solution was passed through a glass column 2.1cm in ID and 50cm long which was filled with 50g of potassium Y type zeolite prepared similarly as in Comparative Example 2. With regard to the elution solvents, first 900mℓ of n-hexane only was passed; then, 2100mℓ of a solution of n-hexane mixed with 0.1% by volume of ethanol was passed. The elution was conducted while holding the column at a temperature of 30°C under normal pressure and at liquid hourly space velocity of the elution solvent of 1.0 (1/hr) and the eluate was divided into 30mℓ fractions. By removing by way of distillation the elution solvent of Fraction Nos. 71 - 74 obtained in this way, 0.18g of fatty acid methyl ester was obtained. The fatty acid composition of this fatty acid esters is shown in Table 5. It indicates that the purity of GLA ester is remarkably upgraded. On the other hand, when a solution of n-hexane mixed with 20% by volume of ethanol was used as the elution solvent, the purity of GLA ester was 70.5%. Example 2 A alga porphyridium cruentum was planted in a culture consisting of 2.7% NaCl, 0.1% KNO₃, 0.007% KH₂PO₄, 0.004% NaHCO₃, 0.66% MgSO₄·7H₂O, 0.56% MgCl₂·6H₂O, 0.15% CaCl₂·2H₂O, 4 × 10⁻⁶% ZnCl₂, 6 × 10⁻⁵% H₃BO₄, 1.5 × 10⁻⁵% CoCl₂·6H₂O, 4 × 10⁻⁶% CuCl₂·2H₂O, 4 × 10⁻⁵% MnCl₂·4H₂O, 3.7 × 10⁻⁵% Mo, 1 mℓ/ℓ of FeCl₂·4H₂O (240 mg/100ml 0.05M Na₂ EDTA) and 20 ml/ℓ of 1M Tris-HCl solution (pH 7.6) and cultivated at a temperature of 23°C and under exposure to approx. 8,000 ℓx illuminance fluorescent light, while passing carbon dioxide gas through the culture. The composition of the lipid obtained in this way which was then, turned into fatty acid methyl esters is shown in Table 6. Five grams of this fatty acid methyl ester mixture was dissolved in 6g n-hexane; with this solution, the same operation as of Example 1 was run and then, by removing the elution solvent of Fraction Nos. 86-90 by way of distillation, 2.3g of fatty acid methyl esters were obtained. The fatty acid composition of this fatty acid esters is shown in Table 7. It suggests that the purity of AA ester is drasticallY improved. Comparative Example 4 Results obtained by the same operation with the same materials as in Comparative Example 1, using various solvents in place of the nonpolar solvent n-heptane and the polar solvent chloroform, are shown in Table 8. The zeolite used was potassium Y type zeolite obtained by the same method as of Comparative Example 1. Results of Table 8 clearly veryfy that in every case, the purity was remarkably improved. Example 3 Mucor ambiguus IFO 6472 was subjected to aerobic cultivation just as in Comparative Example 1 and the lipid accumulated in the fungus was extracted by such a treatment as homogenization. The lipid obtained in this way was subjected to ester exchange, using 0.2N NaOH ethanol solution, yielding an ethyl ester mixture of the fatty acid composition shown in Table 9. Five grams of this fatty acid ethyl ester mixture was dissolved in 5g of n-hexane and this solution was passed through a glass column 1cm in ID and 15cm long filled with 5g of each of various Y type zeolites (24 - 34 mesh) prepared similarly as in Comparative Example 1. As the elution solvent, 2000ml of n-hexane only was passed at first. Then 1000ml of a solution of n-hexane mixed with 0.5% by volume of ethanol was passed. The liquid passing conditions used were the same as of Example 1, but the eluate was divided into 20ml fractions. The elution solvent of Fraction Nos. 11 - 13 obtained in this way were removed by distillation, yielding fatty acid ethyl esters. The fatty acid components and their amounts are shown in Table 10. It suggests that in the case of column operation, very high purity GLA ethyl ester can be obtained using Y type zeolites containing either one of cations - K⁺, Cs⁺, Rb⁺, NH₄⁺, CH₃NH₂⁺, (CH₃)₂NH₂⁺ or C₂H₅NH₃⁺.

Claims: A method of concentrating and separating τ - linolenic acid esters and/or arachidonic acid esters in which their adsorption and desorption are performed by bringing in contact with crystalline aluminosilicate zeolites a fatty acid ester mixture containing τ -linolenic acid esters and/or arachidonic acid esters, characterized in that said crystalline aluminosilicate zeolite has a molar ratio of SiO₂/Al₂O₃, ranging from 3 to 5.3 and that said desorption of τ -linolenic acid esters and/or arachidonic acid esters is performed by treating with a mixture of a polar solvent and a non-polar solvent in proportion of 0.01 - 10 % by volume of the polar solvent to the nonpolar solvent by column operation. The method according to Claim 1, wherein the crystalline aluminosilicate zeolites are zeolites containing at least one cation selected from the group consisting of K⁺, Na⁺, Rb⁺, Cs⁺, NH₄⁺, CH₃NH₃⁺, (CH₃)₂NH₂⁺ and C₂H₅NH₃⁺. The method according to Claim 2, wherein the crystalline aluminosilicate zeolites are zeolites having Na⁺ as a cation at a content of 5 % or lower. The method according to Claim 2 or 3, wherein the crystalline aluminosilicate zeolites containing cations other than Na⁺ are dried at 120°C - 700°C after ion exchange treatment. The method according to Claim 2 or 3, wherein the crystalline aluminosilicate zeolites containing at least one cation selected from the group consisting of NH₄⁺, CH₃NH₃⁺, (CH₃)₂NH₂⁺ and C₂H₅NH₃⁺ are dried at 120°C - 250°C after ion exchange treatment. The method according to Claim 1, wherein the adsorption of the fatty acid esters comprises the following steps of (a) and (b); (a) the fatty acid ester mixture containing τ -linolenic acid esters and/or arachidonic acid esters is dissolved in a nonpolar solvent of 30 times or less by weight of the mixture, and (b) thus obtained solution is passed through a column filled with the aluminosilicate zeolites under a condition of liquid hourly space velocity of 0.5 to 10. The method according to Claim 6, wherein the nonpolar solvent is at least one selected from the group consisting of n-heptane, n-hexane, cyclohexane, benzene, p-xylene and n-octane. The method according to Claim 1, wherein the desorption of the fatty acid esters is performed by passing a polar solvent or a mixture of a nonpolar solvent and a polar solvent through the column under a condition of liquid hourly space velocity of 0.05 - 10. The method according to Claim 1, wherein the polar solvent is at least one selected from the group consisting of acetone, methanol, ethanol, chloroform, diethyl ether and ethyl acetate. The method according to Claim 1, wherein the adsorption and desorption is performed at a temperature from 20°C to 50°C.

Inventors: ARAI, MAKOTO, FUKUDA, HIDEKI
Applicants: KANEGAFUCHI KAGAKU KOGYO KABUSHIKI KAISHA
Application year: 1986
Main classification: C07C67
IPC codes: C07C67, C07C69