TWI648258B - Mothod of purifying polyunsaturated fatty acid and eicosapentaenoic acid - Google Patents
Mothod of purifying polyunsaturated fatty acid and eicosapentaenoic acid Download PDFInfo
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Abstract
一種純化不飽和脂肪酸以及二十碳五烯酸的方法。純化不飽和脂肪酸的方法包括提供乙酯化魚油。以模擬移動床層析法將乙酯化魚油中的包含二十碳五烯酸以及二十二碳六烯酸的不飽和脂肪酸分離開來，藉此得到高純度包含二十碳五烯酸以及二十二碳六烯酸的不飽和脂肪酸。A method of purifying unsaturated fatty acids and eicosapentaenoic acid. A method of purifying an unsaturated fatty acid includes providing an ethyl esterified fish oil. Separating unsaturated fatty acids containing eicosapentaenoic acid and docosahexaenoic acid from ethylated fish oil by simulated moving bed chromatography, thereby obtaining high purity containing eicosapentaenoic acid and An unsaturated fatty acid of docosahexaenoic acid.
Description
本發明是有關於一種純化方法，且特別是有關於一種純化不飽和脂肪酸以及二十碳五烯酸的方法。This invention relates to a purification process, and in particular to a process for the purification of unsaturated fatty acids and eicosapentaenoic acid.
魚油是從魚體內提取的油類物質的統稱，其所含的營養成分對機體有極高的生理活性，是腦、神經組織、骨髓、心、肝、卵和脾中不可缺少的組成部分，同時也有助於脂的消化吸收、轉運和形成，又是生物膜的重要結構物質。Fish oil is a general term for oils extracted from fish. Its nutrients have extremely high physiological activity on the body and are an indispensable component of brain, nerve tissue, bone marrow, heart, liver, egg and spleen. It also contributes to the digestion, absorption, transport and formation of lipids, and is an important structural substance of biofilms.
二十二碳六烯酸（docosahexaenoic acid，DHA）及二十碳五烯酸（eicosapentaenoic acid，EPA）是魚油的主要營養成分。DHA屬於ω-3系列多不飽和脂肪酸，是神經系統細胞生長及維持的一種主要元素，是大腦細胞膜和視網膜的重要構成成分，在人體大腦皮層中含量高達20%，在視網膜中所占比例最大，約占50%〜60% ；它的缺乏可影響大腦智力發育、降低大腦思維、學習、分析及記憶能力，影響大腦分化成熟，影響視力敏感度，減退眼睛視網膜反射力，造成遠視或近視，影響精神集中能力等。Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the main nutrients of fish oil. DHA belongs to the omega-3 series of polyunsaturated fatty acids. It is a major element in the growth and maintenance of the nervous system cells. It is an important component of the brain cell membrane and retina. It is up to 20% in the human cerebral cortex and the largest proportion in the retina. About 50%~60%; its lack can affect brain mental development, reduce brain thinking, learning, analysis and memory, affect brain differentiation and maturity, affect vision sensitivity, reduce eye retinal reflex, cause farsightedness or myopia, Affecting mental concentration and so on.
EPA同樣屬於ω-3系列多不飽和脂肪酸，是人體自身不能合成但又不可缺少的重要營養素，具有幫助降低膽固醇和甘油三酯的含量，促進體內飽和脂肪酸代謝，從而起到降低血液粘稠度、增進血液迴圈、提高組織供氧而消除疲勞、防止脂肪在血管壁的沉積以及預防動脈粥樣硬化的形成和發展、預防腦血栓、腦溢血、高血壓等心血管疾病。EPA is also an omega-3 series of polyunsaturated fatty acids. It is an important nutrient that the human body cannot synthesize but is indispensable. It helps to lower the content of cholesterol and triglycerides, promotes the metabolism of saturated fatty acids in the body, and thus reduces blood viscosity. Improve blood circulation, improve tissue oxygen supply to eliminate fatigue, prevent fat deposition in the blood vessel wall, prevent the formation and development of atherosclerosis, and prevent cardiovascular diseases such as cerebral thrombosis, cerebral hemorrhage, and hypertension.
現有魚油分離純化方法主要有：分子蒸餾法、低溫結晶法、尿素包合法，脂肪酶法、銀樹脂層析法、硝酸銀絡合法和高效液相色譜法。其中，分子蒸餾法、低溫結晶法、尿素包合法、脂肪酶法通常制得的為EPA乙酯（EPA-EE）和DHA乙酯（DHA-EE）的混合物，其單體的純度較低，純度很難達到80%以上。分離純化高純度EPA-EE和DHA-EE 多採用銀樹脂層析法、硝酸銀絡合法和高效液相色譜法。而銀樹脂層析法和硝酸銀絡合法需要使用大量昂貴的硝酸銀，不僅生產成本相對較高，而且硝酸銀難以回收，會造成嚴重污染，且若操作控制不當，硝酸銀還有進入產品的風險。常規的高效液相色譜法很難直接分離純化低純度的魚油原料，難以得到高純度的EPA-EE和DHA-EE，並且由於樣品在純化過程中被高度稀釋。The existing fish oil separation and purification methods mainly include: molecular distillation method, low temperature crystallization method, urea encapsulation method, lipase method, silver resin chromatography method, silver nitrate complex method and high performance liquid chromatography. Among them, a molecular distillation method, a low-temperature crystallization method, a urea encapsulation method, and a lipase method generally produce a mixture of EPA ethyl ester (EPA-EE) and DHA ethyl ester (DHA-EE), and the purity of the monomer is low. The purity is difficult to reach more than 80%. Separation and purification of high-purity EPA-EE and DHA-EE are mostly carried out by silver resin chromatography, silver nitrate complexation and high performance liquid chromatography. Silver resin chromatography and silver nitrate complexation require the use of a large amount of expensive silver nitrate. Not only is the production cost relatively high, but also the silver nitrate is difficult to recycle, which causes serious pollution, and if the operation is improperly controlled, the silver nitrate has a risk of entering the product. Conventional high performance liquid chromatography is difficult to directly separate and purify low-purity fish oil raw materials, and it is difficult to obtain high-purity EPA-EE and DHA-EE, and since the sample is highly diluted in the purification process.
因此，如何找出一種可從魚油中純化出高純度DHA以及EPA的方法，是目前研究人員急欲解決的問題。Therefore, how to find a method for purifying high-purity DHA and EPA from fish oil is an issue that researchers are currently trying to solve.
本發明提供一種純化不飽和脂肪酸的方法，可有效地分離出高純度的包含二十碳五烯酸以及二十二碳六烯酸的不飽和脂肪酸。The present invention provides a method for purifying an unsaturated fatty acid, which is capable of efficiently separating a high-purity unsaturated fatty acid containing eicosapentaenoic acid and docosahexaenoic acid.
本發明提供一種純化二十碳五烯酸的方法，可有效地分離出高純度的二十碳五烯酸。The present invention provides a method for purifying eicosapentaenoic acid, which can effectively separate high-purity eicosapentaenoic acid.
本發明的實施例提供一種純化不飽和脂肪酸的方法。所述方法包括以下步驟。首先，提供乙酯化魚油。接著，以模擬移動床層析法將乙酯化魚油中的不飽和脂肪酸分離開來，其中所分離的不飽和脂肪酸包括二十碳五烯酸以及二十二碳六烯酸，模擬移動床層析法包含：（i）提供模擬移動床，模擬移動床依序包括第一區段、第二區段以及第三區段，其中模擬移動床是由移動相及固定相所組成，固定相顆粒內部是具有孔隙，移動相對於模擬移動床中是朝同一方向從沖滌端入口流經第一區段、第二區段以及第三區段之間，固定相是相對於移動相朝反方向模擬移動，移動相為包含超臨界二氧化碳與純乙醇的沖滌劑；（ii）將乙酯化魚油從進料入口注入模擬移動床的第二區段與第三區段之間，並使不飽和脂肪酸隨固定相移動至第一區段與第二區段之間的萃出端並使乙酯化魚油中的其它混合物隨移動相移動至第三區段的萃餘端，以分離不飽和脂肪酸。Embodiments of the present invention provide a method of purifying an unsaturated fatty acid. The method includes the following steps. First, ethylated fish oil is provided. Next, the unsaturated fatty acids in the ethyl ester fish oil are separated by simulated moving bed chromatography, wherein the isolated unsaturated fatty acids include eicosapentaenoic acid and docosahexaenoic acid, simulating the moving bed. The analysis method comprises: (i) providing a simulated moving bed, the simulated moving bed sequentially comprises a first section, a second section and a third section, wherein the simulated moving bed is composed of a moving phase and a stationary phase, the stationary phase particles The interior has pores, and the movement is in the same direction from the flushing inlet to the first section, the second section and the third section in the same direction, and the stationary phase is opposite to the moving phase. Simulated movement, the mobile phase is a detergent containing supercritical carbon dioxide and pure ethanol; (ii) the ethyl esterified fish oil is injected from the feed inlet between the second section and the third section of the simulated moving bed, and The saturated fatty acid moves with the stationary phase to the extraction end between the first section and the second section and moves the other mixture in the ethyl ester fish oil to the raffinate end of the third section with the mobile phase to separate the unsaturated fatty acid.
在本發明的一實施例中，以沖滌劑的總量計，純乙醇的含量為1 wt%~8 wt%。In an embodiment of the invention, the content of pure ethanol is from 1 wt% to 8 wt% based on the total amount of the detergent.
在本發明的一實施例中，以沖滌劑的總量計，純乙醇的含量為5 wt%。In an embodiment of the invention, the content of pure ethanol is 5 wt% based on the total amount of the detergent.
在本發明的一實施例中，上述的固定相例如是無規二氧化矽。In an embodiment of the invention, the stationary phase is, for example, a random ruthenium dioxide.
在本發明的一實施例中，上述的第一區段、第二區段以及第三區段各自包含2根管柱，且每根管柱內填充顆粒內部具有孔隙的固定相。In an embodiment of the invention, the first section, the second section and the third section each comprise two columns, and each column is filled with a stationary phase having pores inside the particles.
在本發明的一實施例中，上述的模擬移動床使用的分離條件為：二氧化碳流速在沖滌端入口為9.0 克/分鐘、在進料入口為0.64 克/分鐘、在萃出端為4.0 克/分鐘以及在萃餘端為5.64 克/分鐘，且純乙醇流速在沖滌端入口為0.599 毫升/分鐘、在進料入口為0.042 毫升/分鐘、在萃出端為0.268 毫升/分鐘以及在萃餘端為0.378 毫升/分鐘。In an embodiment of the invention, the simulated moving bed is used in a separation condition of: a carbon dioxide flow rate of 9.0 g/min at the flush inlet, 0.64 g/min at the feed inlet, and 4.0 g at the extraction end. /min and at the raffinate end of 5.64 g/min, and the pure ethanol flow rate is 0.599 cc/min at the rinse end inlet, 0.042 cc/min at the feed inlet, and 0.268 cc/min at the extraction end. The remainder is 0.378 ml/min.
在本發明的一實施例中，上述的模擬移動床的切換時間為2分鐘50秒至3分鐘20秒。In an embodiment of the invention, the switching time of the simulated moving bed is 2 minutes 50 seconds to 3 minutes 20 seconds.
在本發明的一實施例中，上述的第一區段、第二區段以及第三區段各自包含2根管柱、3根管柱與3根管柱，且每根管柱內填充顆粒內部具有孔隙的固定相。In an embodiment of the invention, the first segment, the second segment, and the third segment each include two tubular columns, three tubular columns, and three tubular columns, and each of the tubular columns is filled with particles. A stationary phase with pores inside.
在本發明的一實施例中，上述的模擬移動床使用的分離條件為：二氧化碳流速在沖滌端入口為26.5 公斤/小時、在進料入口為1.05 公斤/小時、在萃出端為11.78 公斤/小時以及在萃餘端為15.77 公斤/小時，且純乙醇流速在沖滌端入口為29.39 毫升/分鐘、在進料入口為1.12 毫升/分鐘、在萃出端為13.1毫升/分鐘以及在萃餘端為17.4 毫升/分鐘。In an embodiment of the invention, the simulated moving bed is used under the following conditions: the carbon dioxide flow rate is 26.5 kg/hr at the flush inlet end, 1.05 kg/hr at the feed inlet, and 11.78 kg at the extraction end. /hour and at the raffinate end is 15.77 kg / h, and the pure ethanol flow rate is 29.39 ml / min at the flushing end inlet, 1.12 ml / min at the feed inlet, 13.1 ml / min at the extraction end and in the extraction The remaining end is 17.4 ml/min.
在本發明的一實施例中，上述的模擬移動床的切換時間為4分鐘。In an embodiment of the invention, the switching time of the simulated moving bed is 4 minutes.
本發明的實施例提供一種純化不飽和脂肪酸的方法。所述方法包括以下步驟。首先，提供乙酯化魚油。接著，進行第一模擬移動床層析製程，以將乙酯化魚油中的不飽和脂肪酸分離開來，其中所分離的不飽和脂肪酸包括二十碳五烯酸以及二十二碳六烯酸，其中第一模擬移動床層析製程包含：（i）提供模擬移動床，模擬移動床依序包括第一區段、第二區段以及第三區段，其中模擬移動床是由移動相及固定相所組成，固定相顆粒內部是具有孔隙，移動相對於模擬移動床中是朝同一方向從沖滌端入口流經第一區段、第二區段以及第三區段之間，固定相是相對於移動相朝反方向模擬移動，其中第一模擬移動床層析製程中的移動相為包含超臨界二氧化碳與純乙醇的第一沖滌劑；（ii）將乙酯化魚油從進料入口注入模擬移動床的第二區段與第三區段之間，並使不飽和脂肪酸隨固定相移動至第一區段與第二區段之間的萃出端並使乙酯化魚油中的其它混合物隨移動相移動至第三區段的萃餘端，以分離不飽和脂肪酸。然後，進行第二模擬移動床層析製程，以將所分離的不飽和脂肪酸中的二十碳五烯酸分離開來，其中第二模擬移動床層析製程包括將所分離的不飽和脂肪酸從進料入口注入模擬移動床的第二區段與第三區段之間，並使不飽和脂肪酸中的二十二碳六烯酸隨固定相移動至第一區段與第二區段之間的萃出端並使不飽和脂肪酸中的二十碳五烯酸隨移動相移動至第三區段的萃餘端，以分離二十碳五烯酸以及二十二碳六烯酸，其中第二模擬移動床層析製程中的移動相為包含超臨界二氧化碳與純乙醇的第二沖滌劑。Embodiments of the present invention provide a method of purifying an unsaturated fatty acid. The method includes the following steps. First, ethylated fish oil is provided. Next, a first simulated moving bed chromatography process is performed to separate unsaturated fatty acids in the ethylated fish oil, wherein the isolated unsaturated fatty acids include eicosapentaenoic acid and docosahexaenoic acid. The first simulated moving bed chromatography process comprises: (i) providing a simulated moving bed, the simulated moving bed sequentially comprising a first section, a second section and a third section, wherein the simulated moving bed is moved by the mobile phase and fixed The phase consists of the inside of the stationary phase particles having pores moving in the same direction from the flushing end inlet through the first section, the second section and the third section in the same direction as the simulated moving bed, the stationary phase is Simulating movement in the opposite direction relative to the mobile phase, wherein the mobile phase in the first simulated moving bed chromatography process is a first detergent containing supercritical carbon dioxide and pure ethanol; (ii) the ethyl esterified fish oil is fed from the inlet Injecting between the second section and the third section of the simulated moving bed, and moving the unsaturated fatty acid with the stationary phase to the extraction end between the first section and the second section and causing the esterification in the fish oil Other mixtures move with Raffinate to move the end of the third section, to separate unsaturated fatty acids. Then, performing a second simulated moving bed chromatography process to separate the eicosapentaenoic acid in the separated unsaturated fatty acid, wherein the second simulated moving bed chromatography process comprises separating the separated unsaturated fatty acids from The feed inlet is injected between the second section and the third section of the simulated moving bed, and the docosahexaenoic acid in the unsaturated fatty acid is moved with the stationary phase to between the first section and the second section The extraction end and the eicosapentaenoic acid in the unsaturated fatty acid is moved to the raffinate end of the third segment with the mobile phase to separate eicosapentaenoic acid and docosahexaenoic acid, wherein The mobile phase in the two simulated moving bed chromatography process is a second detergent containing supercritical carbon dioxide and pure ethanol.
在本發明的一實施例中，以第一沖滌劑的總量計，純乙醇的含量為1 wt%~8 wt%。In an embodiment of the invention, the content of pure ethanol is from 1 wt% to 8 wt% based on the total amount of the first detergent.
在本發明的一實施例中，以第一沖滌劑的總量計，純乙醇的含量為5 wt%。In an embodiment of the invention, the content of pure ethanol is 5 wt% based on the total amount of the first detergent.
在本發明的一實施例中，以第二沖滌劑的總量計，純乙醇的含量為1 wt%~8 wt%。In an embodiment of the invention, the content of pure ethanol is from 1 wt% to 8 wt% based on the total amount of the second detergent.
在本發明的一實施例中，以第二沖滌劑的總量計，純乙醇的含量為2.5 wt%。In an embodiment of the invention, the content of pure ethanol is 2.5% by weight based on the total amount of the second detergent.
在本發明的一實施例中，上述的固定相例如是無規二氧化矽。In an embodiment of the invention, the stationary phase is, for example, a random ruthenium dioxide.
在本發明的一實施例中，上述的第一區段、第二區段以及第三區段各自包含2根管柱、3根管柱與3根管柱，且每根管柱內填充顆粒內部具有孔隙的固定相。In an embodiment of the invention, the first segment, the second segment, and the third segment each include two tubular columns, three tubular columns, and three tubular columns, and each of the tubular columns is filled with particles. A stationary phase with pores inside.
在本發明的一實施例中，上述的第一模擬移動床層析製程的分離條件為：二氧化碳流速在沖滌端入口為26.5 公斤/小時、在進料入口為1.05 公斤/小時、在萃出端為11.78 公斤/小時以及在萃餘端為15.77 公斤/小時，且純乙醇流速在沖滌端入口為29.39 毫升/分鐘、在進料入口為1.12 毫升/分鐘、在萃出端為13.1毫升/分鐘以及在萃餘端為17.4 毫升/分鐘，且模擬移動床的切換時間為4分鐘。In an embodiment of the invention, the separation condition of the first simulated moving bed chromatography process is: the carbon dioxide flow rate is 26.5 kg/hr at the inlet of the flushing end, and 1.05 kg/hr at the inlet of the feed, in the extraction. The end was 11.78 kg/hr and the raffinate end was 15.77 kg/hr, and the pure ethanol flow rate was 29.39 ml/min at the flush inlet, 1.12 ml/min at the feed inlet, and 13.1 ml at the extraction end. The minute and the raffinate end were 17.4 ml/min, and the switching time of the simulated moving bed was 4 minutes.
在本發明的一實施例中，第二模擬移動床層析製程的分離條件為：二氧化碳流速在沖滌端入口為26.5 公斤/小時、在進料入口為0.3 公斤/小時、在萃出端為11.78 公斤/小時以及在萃餘端為15.02 公斤/小時，且純乙醇流速在沖滌端入口為14.7 毫升/分鐘、在進料入口為0.165 毫升/分鐘、在萃出端為6.55毫升/分鐘以及在萃餘端為8.315 毫升/分鐘，且模擬移動床的切換時間為5分鐘15秒至5分鐘40秒。In an embodiment of the invention, the separation condition of the second simulated moving bed chromatography process is: the carbon dioxide flow rate is 26.5 kg/hr at the inlet of the flushing end, 0.3 kg/hr at the inlet of the feed, and at the extraction end. 11.78 kg/h and 15.02 kg/hr at the raffinate end, and the pure ethanol flow rate was 14.7 ml/min at the flush inlet, 0.165 ml/min at the feed inlet, and 6.55 ml/min at the extraction end. At the raffinate end, it is 8.315 ml/min, and the switching time of the simulated moving bed is 5 minutes 15 seconds to 5 minutes 40 seconds.
基於上述，本發明的不飽和脂肪酸的純化方法透過應用模擬移動床層析法來從魚油中分離出包含EPA以及DHA的不飽和脂肪酸，不僅可有效提升分離效率，更可獲得高純度的包含EPA以及DHA的不飽和脂肪酸。此外，本發明的二十碳五烯酸的純化方法可藉由進行二次模擬移動床層析製程而進一步從魚油中純化出二十碳五烯酸，同樣地，不僅可有效提升分離效率，更可獲得高純度的二十碳五烯酸。Based on the above, the method for purifying the unsaturated fatty acid of the present invention separates the unsaturated fatty acid containing EPA and DHA from the fish oil by using simulated moving bed chromatography, which not only can effectively improve the separation efficiency, but also can obtain high-purity EPA. And unsaturated fatty acids of DHA. In addition, the purification method of eicosapentaenoic acid of the present invention can further purify eicosapentaenoic acid from fish oil by performing a secondary simulated moving bed chromatography process, and similarly, not only can the separation efficiency be effectively improved, More high-purity eicosapentaenoic acid is obtained.
為讓本發明的上述特徵和優點能更明顯易懂，下文特舉實施例，並配合所附圖式作詳細說明如下。The above described features and advantages of the invention will be apparent from the following description.
本發明實施例的純化EPA的方法，是可用以將EPA以及其它混合物從魚油中分離純化出來的方法。藉此，能夠得到高純度的EPA。The method of purifying EPA according to an embodiment of the present invention is a method which can be used to separate and purify EPA and other mixtures from fish oil. Thereby, high purity EPA can be obtained.
以下列舉實施例以說明本發明純化方法之細節或條件，並且下述實施例主要分成兩大部分。第一部分是關於魚油中不飽脂肪酸的純化，更具體來說，是關於魚油中包含EPA以及DHA的不飽脂肪酸的純化。純化不飽脂肪酸的方法包括：提供乙酯化魚油；以及以模擬移動床層析法將乙酯化魚油中的不飽和脂肪酸分離開來，其中所分離的不飽和脂肪酸包括二十碳五烯酸以及二十二碳六烯酸。The following examples are given to illustrate the details or conditions of the purification process of the present invention, and the following examples are largely divided into two major parts. The first part is about the purification of unsaturated fatty acids in fish oil, more specifically, the purification of unsaturated fatty acids containing EPA and DHA in fish oil. The method for purifying unsaturated fatty acids comprises: providing ethylated fish oil; and separating unsaturated fatty acids in the ethylated fish oil by simulated moving bed chromatography, wherein the isolated unsaturated fatty acids include eicosapentaenoic acid And docosahexaenoic acid.
第二部分是關於魚油中EPA的純化，更具體來說，是先將包含EPA以及DHA的不飽脂肪酸從魚油中分離，再將EPA從包含EPA以及DHA的不飽脂肪酸中分離。純化EPA的方法包括：提供乙酯化魚油；進行第一模擬移動床層析製程，以將乙酯化魚油中的不飽和脂肪酸分離開來，其中所分離的不飽和脂肪酸包括二十碳五烯酸（eicosapentaenoic acid，EPA）以及二十二碳六烯酸（docosahexaenoic acid，DHA）；以及進行第二模擬移動床層析製程，以將所分離的不飽和脂肪酸中的二十碳五烯酸以及二十二碳六烯酸分離開來。The second part is about the purification of EPA in fish oil, more specifically, the separation of unsaturated fatty acids containing EPA and DHA from fish oil, and then separating EPA from unsaturated fatty acids containing EPA and DHA. The method for purifying EPA comprises: providing ethylated fish oil; performing a first simulated moving bed chromatography process to separate unsaturated fatty acids in ethylated fish oil, wherein the isolated unsaturated fatty acid comprises eicosapentaene Acid (eicosapentaenoic acid, EPA) and docosahexaenoic acid (DHA); and performing a second simulated moving bed chromatography process to remove eicosapentaenoic acid from the isolated unsaturated fatty acids and The docosahexaenoic acid is separated.
以下的實施例非用以限制本發明保護範圍。所繪圖式係為示意圖僅為說明方便而繪製，並非代表限制其實際之方法、條件或裝置等。 實施例1： 不飽脂肪酸的純化 The following examples are not intended to limit the scope of the invention. The drawings are schematic for the convenience of description and are not intended to limit the actual methods, conditions, or devices. Example 1: Purification of unsaturated fatty acids
在本實施例中，可使用如圖1所示的超臨界流體模擬移動床（Supercritical Fluid-Simulated Moving Bed，SF-SMB）設備來進行模擬移動床層析法。圖1是依照本發明實施例的一種超臨界流體模擬移動床設備的管線流程圖。請參照圖1，模擬移動床100包括第一區段、第二區段與第三區段。在本實施例中，第一區段包含2根管柱C1與C2，第二區段包含3根管柱C3、C4與C5，且第三區段包含3根管柱C6、C7與C8，上述8根管柱串聯，但本發明不限於此。在另一實施例中，第一區段包含2根管柱，第二區段包含2根管柱，且第三區段包含2根管柱，上述6根管柱串聯。In the present embodiment, a simulated moving bed chromatography method can be performed using a Supercritical Fluid-Simulated Moving Bed (SF-SMB) apparatus as shown in FIG. 1 is a pipeline flow diagram of a supercritical fluid simulated moving bed apparatus in accordance with an embodiment of the present invention. Referring to FIG. 1, the simulated moving bed 100 includes a first section, a second section, and a third section. In this embodiment, the first section comprises two columns C1 and C2, the second section comprises three columns C3, C4 and C5, and the third section comprises three columns C6, C7 and C8, The above eight columns are connected in series, but the invention is not limited thereto. In another embodiment, the first section comprises 2 columns, the second section comprises 2 columns, and the third section comprises 2 columns, said 6 columns being connected in series.
模擬移動床100是由移動相（未繪示）及固定相（未繪示）所組成。移動相是相對於模擬移動床100中是朝同一方向從沖滌端入口D1流經第一區段、第二區段以及第三區段之間，而固定相是相對於移動相朝反方向模擬移動。The simulated moving bed 100 is composed of a mobile phase (not shown) and a stationary phase (not shown). The moving phase is flowing from the flushing end inlet D1 through the first section, the second section and the third section in the same direction relative to the simulated moving bed 100, and the stationary phase is opposite to the moving phase Simulate movement.
每根管柱內填充有顆粒內部具有孔隙的固定相。在本實施例中，固定相例如是無規二氧化矽（irregular silica）。但本發明不限於此，固定相可以為習知常用的固定相材料。在本實施例中，移動相（或沖滌劑）例如是包含超臨界二氧化碳與輔助溶劑的沖滌劑。在本實施例中，輔助溶劑為純乙醇（無水乙醇）。包含超臨界二氧化碳與輔助溶劑的沖滌劑可藉由二氧化碳液泵產生高壓二氧化碳並與輔助溶劑混合後而形成。Each column is filled with a stationary phase having pores inside the particles. In this embodiment, the stationary phase is, for example, random silica. However, the invention is not limited thereto, and the stationary phase may be a conventionally used stationary phase material. In this embodiment, the mobile phase (or detergent) is, for example, a detergent comprising supercritical carbon dioxide and an auxiliary solvent. In this embodiment, the auxiliary solvent is pure ethanol (anhydrous ethanol). A detergent containing supercritical carbon dioxide and an auxiliary solvent can be formed by a high-pressure carbon dioxide generated by a carbon dioxide liquid pump and mixed with an auxiliary solvent.
再次參照圖1，模擬移動床100包括兩個入料口，分別為樣品進料入口F1（即管柱C6入口位置）與沖滌端入口D1（即管柱C1入口位置），且包括兩個出料口，分別為萃出端E1（即管柱C2出口位置）與萃餘端R1（即管柱C8出口位置）。如果讓所有入料口以及出料口的位置在經過一段時間後，同時轉換至下一支管柱，則可模擬固定相移動（即向圖1的下方移動）。舉例來說，進料入口由原來在管柱C6入口位置切換至管柱C7入口位置，其餘的入料口以及出料口亦同時往下一支管柱變換，在此同時，沖滌劑與進料則仍然一直連續不斷地往萃餘端流動。如果不斷地連續切換進料口以及出料口的位置，則會形成讓固體連續向下流動並一再循環，因此可達成固體與超臨界流體連續逆向流動接觸的過程。Referring again to Figure 1, the simulated moving bed 100 includes two inlets, a sample feed inlet F1 (i.e., the inlet position of the column C6) and a flush inlet end D1 (i.e., the inlet position of the column C1), and includes two The discharge port is the extraction end E1 (ie, the outlet position of the column C2) and the ripping end R1 (ie, the outlet position of the column C8). If all the inlets and outlets are moved to the next column after a period of time, the stationary phase movement can be simulated (ie moving to the lower side of Figure 1). For example, the feed inlet is switched from the inlet position of the column C6 to the inlet position of the column C7, and the other inlet and outlet are simultaneously changed to the next column. At the same time, the detergent and the inlet are simultaneously The material is still flowing continuously to the end of the collection. If the feed port and the position of the discharge port are continuously switched continuously, a solid is continuously flowed downward and recirculated, so that a continuous reverse flow contact of the solid with the supercritical fluid can be achieved.
由於本發明實施例是使用超臨界二氧化碳作為沖滌劑（移動相），因此需要設置一個高壓的二氧化碳供應源110。模擬移動床100是利用二氧化碳液泵115從二氧化碳供應源110產生高壓二氧化碳，並暫存於高壓緩衝槽120之中。接著，再以前端壓力調壓閥122或後端壓力調壓閥123、質量流量計並搭配控制閥（未繪示）來控制進料的二氧化碳流速。Since the embodiment of the present invention uses supercritical carbon dioxide as a detergent (mobile phase), it is necessary to provide a high-pressure carbon dioxide supply source 110. The simulated moving bed 100 generates high-pressure carbon dioxide from the carbon dioxide supply source 110 by the carbon dioxide liquid pump 115 and temporarily stores it in the high-pressure buffer tank 120. Then, the front end pressure regulating valve 122 or the rear pressure regulating valve 123, the mass flow meter and the control valve (not shown) are used to control the carbon dioxide flow rate of the feed.
除了二氧化碳質量流量的控制以外，輔助溶劑的輸入則從輸入口D2藉由高效能液相層析液泵125a加以控制，而樣品的輸入則從輸入口F2藉由高效能液相層析液泵125b加以控制。詳細來說，待樣品的進料溶解於輔助溶劑中後，其是利用高效能液相層析液泵125b從輸入口F2輸入與二氧化碳混合後再進入模擬移動床100中。相同地，做為移動相，包含超臨界二氧化碳以及輔助溶劑的沖滌液是藉由二氧化碳液泵115產生的高壓二氧化碳與從輸入口D2輸入的輔助溶劑混合後而形成。此外，上述的高壓二氧化碳與輔助溶劑混合的步驟可藉由混合器130來達成。In addition to the control of the mass flow rate of carbon dioxide, the input of the auxiliary solvent is controlled from the input port D2 by the high performance liquid chromatography pump 125a, and the input of the sample is from the input port F2 by the high performance liquid chromatography pump. 125b is controlled. In detail, after the sample to be sample is dissolved in the auxiliary solvent, it is mixed with carbon dioxide from the input port F2 by the high performance liquid chromatography pump 125b and then enters the simulated moving bed 100. Similarly, as the mobile phase, the rinse liquid containing supercritical carbon dioxide and the auxiliary solvent is formed by mixing the high pressure carbon dioxide generated by the carbon dioxide liquid pump 115 with the auxiliary solvent input from the input port D2. Further, the above step of mixing the high pressure carbon dioxide with the auxiliary solvent can be achieved by the mixer 130.
超臨界流體在連續切換進料口以及出料口位置的同時，雖然超臨界流體不斷向上流動（即向圖1的上方移動），但是並沒有直接循環回到管柱C1位置。傳統以液體為流動相的模擬移動床裝置，經常會增設第四區段，用以再生流動相進而直接循環回流使用。在本實施例中，使用降壓分離方式而輕易達成超臨界流體的再生，因此從萃餘端R1以及萃出端E1流出的超臨界流體，經過分離槽145a、145b的簡單降壓後將二氧化碳汽化，便可將二氧化碳氣體經過二次冷卻沉澱出殘留的輔助溶劑與溶質後達成二氧化碳再生之目的。如此便可以減少第四區段的管柱使用、降低設備的成本以及填料需求的成本。While the supercritical fluid continuously switches the feed port and the discharge port position, although the supercritical fluid continuously flows upward (ie, moves upward in FIG. 1), it does not directly circulate back to the column C1 position. Conventionally, a moving moving bed device using a liquid as a mobile phase often has a fourth section for regenerating the mobile phase and directly recycling it. In the present embodiment, the regeneration of the supercritical fluid is easily achieved using the pressure reduction separation mode, so that the supercritical fluid flowing out from the rugged end R1 and the extraction end E1 passes through the simple depressurization of the separation grooves 145a, 145b to carbon dioxide. By vaporization, the carbon dioxide gas can be cooled by secondary cooling to precipitate residual auxiliary solvent and solute for carbon dioxide regeneration. This can reduce the use of the column in the fourth section, reduce the cost of the equipment, and the cost of the packing requirements.
由分離槽155回收的二氧化碳氣體經過冷凝回收以後，暫存於工作儲槽160，再經預冷後以二氧化碳液泵115加壓暫存於高壓緩衝槽120中，並以後端壓力調壓閥123控制其壓力。高壓緩衝槽120內的二氧化碳經過適度的調壓與計量後，分別由管柱C1與管柱C6位置注入到系統之中，注入之前並與定量輸入的輔助溶劑或是進料溶液混合。經過模擬移動床的分離作用之後，兩個出料則由萃出端E1與萃餘端R1流出系統外。萃餘端R1流出的超臨界流體先經過後端壓力調壓閥123後在分離槽145b中分離出輔助溶劑與溶質，然後回收二氧化碳氣體。在萃餘端R1出口的後端壓力調壓閥123也負責控制著整個SF-SMB的操作壓力。萃出端E1流出的超臨界流體則藉由一個質量流量控制閥控制其流出的流速，然後進入分離槽145a分離出輔助溶劑與溶質。從萃出端E1與萃餘端R1分離槽流出的二氧化碳氣體合併後再一起循環回收。The carbon dioxide gas recovered by the separation tank 155 is condensed and recovered, temporarily stored in the working storage tank 160, and then pre-cooled, and then temporarily pressurized by the carbon dioxide liquid pump 115 in the high-pressure buffer tank 120, and the pressure regulating valve 123 at the rear end. Control its pressure. After a moderate pressure regulation and metering, the carbon dioxide in the high-pressure buffer tank 120 is injected into the system from the column C1 and the column C6, respectively, and mixed with the auxiliary solvent or the feed solution. After the separation of the simulated moving bed, the two discharges flow out of the system from the extraction end E1 and the raffinate end R1. The supercritical fluid flowing out of the rugged end R1 passes through the back pressure regulating valve 123, and then separates the auxiliary solvent and the solute in the separating tank 145b, and then recovers the carbon dioxide gas. The rear pressure regulating valve 123 at the outlet of the rugged end R1 is also responsible for controlling the operating pressure of the entire SF-SMB. The supercritical fluid flowing out of the extraction end E1 controls the flow rate of the outflow by a mass flow control valve, and then enters the separation tank 145a to separate the auxiliary solvent and the solute. The carbon dioxide gas flowing out from the separation end E1 and the raffinate end R1 separation tank is combined and recycled together.
接著，以下將對利用模擬移動床層析法將包含EPA以及DHA的不飽脂肪酸從魚油中分離開來的方式進行說明。在提供如圖1所示的模擬移動床100之後，是將乙酯化魚油從進料入口F1注入模擬移動床100的第二區段以及第三區段之間，並且使包含EPA以及DHA的不飽脂肪酸隨固定相移動至第一區段與第二區段之間的萃出端E1並使乙酯化魚油中的其它混合物隨移動相移動至第三區段的萃餘端R1。為了達到上述的分離結果，移動相選擇包含超臨界二氧化碳與純乙醇的沖滌劑。在本實施例中，以沖滌劑的總量計，純乙醇的含量為1 wt%~8 wt%。在一實施例中，以沖滌劑的總量計，純乙醇的含量為5%。 分析方法建立 Next, the manner in which the unsaturated fatty acid containing EPA and DHA is separated from fish oil by simulated moving bed chromatography will be described below. After providing the simulated moving bed 100 as shown in FIG. 1, the ethyl esterified fish oil is injected from the feed inlet F1 between the second section and the third section of the simulated moving bed 100, and the EPA and DHA are included. The unsaturated fatty acid moves with the stationary phase to the extraction end E1 between the first section and the second section and causes the other mixture in the esterified fish oil to move with the mobile phase to the raffinate end R1 of the third section. In order to achieve the above separation results, the mobile phase selects a detergent containing supercritical carbon dioxide and pure ethanol. In the present embodiment, the content of pure ethanol is from 1 wt% to 8 wt% based on the total amount of the detergent. In one embodiment, the pure ethanol is present in an amount of 5% based on the total amount of the detergent. Analytical method establishment
分析方法中是使用安捷倫氣相層析質譜儀（GC/MS）（型號7890A/59770B）進行乙酯化魚油的分析，所使用的分析毛細管柱為DB-5MS (30 m× 250 μm)，並選用1.0毫升/分鐘氦氣作為挾帶氣體。氣相層析質譜儀的升溫條件設定如下：起始120℃並以10℃/分鐘升溫至220℃後持溫6分鐘，再以10℃/分鐘升溫至250℃持溫12分鐘，再以5℃/分鐘升溫至300℃持溫5分鐘，進樣量為1μL，採用分流30:1。Analytical method was performed using an Agilent gas chromatography mass spectrometer (GC/MS) (model 7890A/59770B) for the analysis of ethyl ester fish oil using an analytical capillary column of DB-5MS (30 m × 250 μm). 1.0 ml/min of helium is used as the gas. The temperature rising condition of the gas chromatography mass spectrometer was set as follows: starting at 120 ° C and raising the temperature to 220 ° C at 10 ° C / minute, holding the temperature for 6 minutes, and then raising the temperature to 250 ° C at 10 ° C / minute for 12 minutes, and then 5 The temperature was raised to 300 ° C for 5 minutes at ° C / min, the injection volume was 1 μL, and the split flow was 30:1.
樣品的GC/MS圖譜如圖2與圖3所示。圖2為10%乙酯化魚油的氣相色譜質譜分析圖。圖3為25%乙酯化魚油的氣相色譜質譜分析圖。在圖2與圖3中，內標準品IS採用167毫克/升的十五烷。依據MS資料庫數據比對，在滯留時間為17.5 分鐘處為EPA，而在滯留時間為20.5分鐘處為DHA。從GC/MS圖譜可以清楚判讀出DHA、EPA以及其它混合物102的波鋒位置，藉由此結果做為分析標準。 [SF-SMB 分離試驗 ] [ 包含 EPA 與 DHA 的不飽和脂肪酸 的純度以及回收率計算 ] The GC/MS spectra of the samples are shown in Figures 2 and 3. Figure 2 is a gas chromatographic mass spectrogram of 10% ethyl ester fish oil. Figure 3 is a gas chromatographic mass spectrogram of 25% ethyl esterified fish oil. In Figures 2 and 3, the internal standard IS employs 167 mg/l of pentadecane. According to the MS database data comparison, the EPA was at a residence time of 17.5 minutes, and the DHA was at a residence time of 20.5 minutes. The position of the wave front of DHA, EPA, and other mixtures 102 can be clearly discerned from the GC/MS spectrum, and the results are used as analytical standards. [SF-SMB separation test] [containing EPA and DHA unsaturated fatty acid purity and recovery rate calculation]
首先，將乙酯化魚油樣品直接溶解於純乙醇當作進料溶液。然後，再利用液泵輸入SF-SMB系統以分離成兩種不同組成的樣品。由萃出端E1取樣得到的樣品需進行其中EPA與DHA的含量計算，並與進料入口F1中EPA與DHA的含量相比較，以判定分離的效果。從萃出端E1得到的樣品中量取一定體積的樣品後，先稀釋10倍，然後按照體積比為9:1的比例與已知濃度的內標物均勻混和後，以GC/MS分析得圖譜中EPA與DHA的面積分率，因此EPA與DHA的含量計算公式如下式1所示。在本實施例中，EPA與DHA的含量可定義為純度。 (式1) 在式1中，A EPA與A DHA分別為GC/MS圖譜中EPA與DHA的峰面積，SA i表示圖譜中所有的峰面積總和。 First, the ethyl esterified fish oil sample was directly dissolved in pure ethanol as a feed solution. Then, the liquid pump was used to input the SF-SMB system to separate into two samples of different compositions. The sample sampled by the extraction end E1 is subjected to calculation of the content of EPA and DHA, and compared with the content of EPA and DHA in the feed inlet F1 to determine the effect of separation. After taking a certain volume of the sample from the sample at the extraction end E1, it is diluted 10 times, and then uniformly mixed with a known concentration of the internal standard according to a volume ratio of 9:1, and then analyzed by GC/MS. The area fraction of EPA and DHA in the map, so the formula for calculating the content of EPA and DHA is as shown in the following formula 1. In this embodiment, the content of EPA and DHA can be defined as purity. (Formula 1) In Formula 1, A EPA and A DHA are the peak areas of EPA and DHA in the GC/MS spectrum, respectively, and SA i represents the sum of all peak areas in the spectrum.
EPA與DHA從乙酯化魚油中分離以後，其回收率則依以下式2來估算。 (式2) 在式2中，A EPA與A DHA分別為GC/MS圖譜中EPA與DHA的峰面積，上標E與R分別代表萃出端以及萃餘端，而Q EtOH為乙醇的體積流速。 實驗例 1 [SF-SMB 的操作條件 ] After the separation of EPA and DHA from ethyl ester fish oil, the recovery rate is estimated according to the following formula 2. (Formula 2) In Equation 2, A EPA and A DHA are the peak areas of EPA and DHA in the GC/MS spectrum, respectively. Superscripts E and R represent the extraction end and the raffinate end, respectively, and Q EtOH is the volume of ethanol. Flow rate. Experimental Example 1 [ Operation conditions of SF-SMB ]
在實驗例1中，10%乙酯化魚油原料（常州嘉眾新材料科技有限公司）先配製成20 克/升的乙醇溶液。接著，使用類似於圖1所示的超臨界流體模擬移動床設備來進行包含EPA與DHA的不飽和脂肪酸的純化。與圖1的超臨界流體模擬移動床設備的差別僅在於在實驗例1中是使用6根填充管柱被設計成三個區段的模擬移動床。填充管柱為10 mm × 150 mm的不銹鋼管柱，所採用的填料（固定相）為無規二氧化矽，而移動相為包含超臨界二氧化碳與5wt%純乙醇的沖滌劑。6根填充管柱被設計成三個區段的模擬移動床，而且每一個區段各有2支管柱。分離的條件為：溫度固定為50℃，萃餘端出口壓力為1700 psi，而沖滌劑入口壓力2200 psi。各入口與出口端的二氧化碳流速設定如下：沖滌端入口為9.0 克/分鐘；進料入口為0.64 克/分鐘；萃出端為4.0 克/分鐘；萃餘端為5.64 克/分鐘（經質量守恆計算）。入料口的純乙醇流速設定如下：沖滌端入口為0.599 毫升/分鐘；進料入口為0.042 毫升/分鐘。依據質量守恆，萃出端與萃餘端的純乙醇流速分別為0.268毫升/分鐘以及0.378 毫升/分鐘。此外，在實驗例1中，在固定各出入口的流速條件下，改變SF-SMB設備上閥門的切換時間（2分鐘40秒、2分鐘50秒與3分鐘），然後觀察二個出料口所收集樣品的組成隨切換時間的變化。採用上述條件進行模擬移動床層析法所得到的結果分析如圖4至圖6所示，且由式1與式2所計算含量（定義為純度）與回收率的結果如表1所示。In Experimental Example 1, 10% ethyl ester fish oil raw material (Changzhou Jiazhong New Material Technology Co., Ltd.) was first formulated into a 20 g/L ethanol solution. Next, purification of the unsaturated fatty acid containing EPA and DHA was carried out using a supercritical fluid simulated moving bed apparatus similar to that shown in FIG. The difference from the supercritical fluid simulated moving bed apparatus of Fig. 1 is only that in the experimental example 1, a simulated moving bed designed to be three sections using six packed columns is used. The packed column is a 10 mm × 150 mm stainless steel column with a filler (stationary phase) of random cerium oxide and a mobile phase of a detergent containing supercritical carbon dioxide and 5 wt% pure ethanol. The six packed columns are designed as three sections of simulated moving bed with two columns each. Separation conditions were such that the temperature was fixed at 50 ° C, the raffinate outlet pressure was 1700 psi, and the detergent inlet pressure was 2200 psi. The carbon dioxide flow rates at each inlet and outlet were set as follows: 9.0 g/min for the flush inlet; 0.64 g/min for the feed inlet; 4.0 g/min for the extraction end; 5.64 g/min for the raffinate end (conservation of mass) Calculation). The pure ethanol flow rate at the feed port was set as follows: the flush inlet inlet was 0.599 cc/min; the feed inlet was 0.042 cc/min. According to the conservation of mass, the pure ethanol flow rates at the extraction end and the raffinate end were 0.268 ml/min and 0.378 ml/min, respectively. Further, in Experimental Example 1, the switching time of the valve on the SF-SMB device was changed (2 minutes, 40 seconds, 2 minutes, 50 seconds, and 3 minutes) under the condition of fixing the flow rate of each inlet and outlet, and then the two discharge ports were observed. The composition of the collected samples varies with the switching time. The results obtained by the simulated moving bed chromatography using the above conditions were analyzed as shown in Figs. 4 to 6, and the results (defined as purity) and recovery rates calculated by the formulas 1 and 2 are shown in Table 1.
表1 <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> 純度(EPA+DHA) (%) </td><td> 回收率 (%) </td></tr><tr><td> 進料入口 </td><td> 65.8 </td><td> </td></tr><tr><td> 切換時間 2分鐘40秒 </td><td> 65.9 </td><td> 100 </td></tr><tr><td> 切換時間 2分鐘50秒 </td><td> 88.7 </td><td> 83.3 </td></tr><tr><td> 切換時間 3分鐘 </td><td> 92 </td><td> 83.3 </td></tr></TBODY></TABLE>Table 1 <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> Purity (EPA+DHA) (%) </td>< Td> recovery rate (%) </td></tr><tr><td> feed inlet</td><td> 65.8 </td><td> </td></tr><tr> <td> Switching time 2 minutes 40 seconds</td><td> 65.9 </td><td> 100 </td></tr><tr><td> Switching time 2 minutes 50 seconds</td>< Td> 88.7 </td><td> 83.3 </td></tr><tr><td> Switching time 3 minutes</td><td> 92 </td><td> 83.3 </td>< /tr></TBODY></TABLE>
圖4至圖6為本發明實驗例利用模擬移動床層析法從10%魚油中分離純化出包含EPA以及DHA的不飽和脂肪酸的結果分析圖。請參照圖4至圖6以及表1。由圖4可以看出，在切換時間為2分鐘40秒時，並無法將包含DHA以及EPA的不飽和脂肪酸由魚油中分離純化出來。而由圖5以及圖6可以看出，包含DHA以及EPA的不飽和脂肪酸是從萃出端E1分離出來，而大部分的其它混合物102是從萃餘端R1分離出來。且在切換時間為3分鐘時，包含DHA以及EPA的不飽和脂肪酸的純度可由原本的65.8%提高至92%。 實驗例 2 4 to 6 are graphs showing the results of separation and purification of unsaturated fatty acids containing EPA and DHA from 10% fish oil by simulated moving bed chromatography in the experimental example of the present invention. Please refer to FIG. 4 to FIG. 6 and Table 1. As can be seen from Fig. 4, when the switching time is 2 minutes and 40 seconds, the unsaturated fatty acid containing DHA and EPA cannot be separated and purified from fish oil. As can be seen from Fig. 5 and Fig. 6, the unsaturated fatty acid containing DHA and EPA is separated from the extraction end E1, and most of the other mixture 102 is separated from the raffinate end R1. And when the switching time is 3 minutes, the purity of the unsaturated fatty acid containing DHA and EPA can be increased from 65.8% to 92%. Experimental example 2
採用與實驗例1相同的設備和條件來進行純化，其差別僅在於將25%乙酯化魚油原料製成10 克/升的乙醇溶液，以及進行五種不同切換時間（2分鐘50秒、2分鐘55秒、3分鐘、3分鐘20秒以及3分鐘40秒）的試驗。採用上述條件進行模擬移動床層析法所得到的結果分析如圖7至圖11所示，且由式1與式2所計算含量（定義為純度）與回收率的結果如表2所示。Purification was carried out using the same equipment and conditions as in Experimental Example 1, except that the 25% ethyl ester fish oil raw material was made into a 10 g/L ethanol solution, and five different switching times were performed (2 minutes 50 seconds, 2 Tests of 55 seconds, 3 minutes, 3 minutes 20 seconds, and 3 minutes 40 seconds). The results obtained by the simulated moving bed chromatography using the above conditions were analyzed as shown in Figs. 7 to 11, and the results of the contents (defined as purity) and the recovery ratios calculated by the formulas 1 and 2 are shown in Table 2.
表2 <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> 純度(EPA+DHA) (%) </td><td> 回收率 (%) </td></tr><tr><td> 進料入口 </td><td> 55.3 </td><td> </td></tr><tr><td> 切換時間 2分鐘50秒 </td><td> 85.5 </td><td> 96.5 </td></tr><tr><td> 切換時間 2分鐘55秒 </td><td> 86.8 </td><td> 95.6 </td></tr><tr><td> 切換時間 3分鐘 </td><td> 87.2 </td><td> 93.8 </td></tr><tr><td> 切換時間 3分鐘20秒 </td><td> 89.3 </td><td> 39.9 </td></tr></TBODY></TABLE>Table 2 <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> Purity (EPA+DHA) (%) </td>< Td> recovery rate (%) </td></tr><tr><td> feed inlet</td><td> 55.3 </td><td> </td></tr><tr> <td> Switching time 2 minutes 50 seconds</td><td> 85.5 </td><td> 96.5 </td></tr><tr><td> Switching time 2 minutes 55 seconds</td>< Td> 86.8 </td><td> 95.6 </td></tr><tr><td> Switching time 3 minutes</td><td> 87.2 </td><td> 93.8 </td>< /tr><tr><td> Switching time 3 minutes 20 seconds</td><td> 89.3 </td><td> 39.9 </td></tr></TBODY></TABLE>
圖7至圖11為本發明實驗例利用模擬移動床層析法從25%魚油中分離純化出包含EPA以及DHA的不飽和脂肪酸的結果分析圖。請參照圖7至圖11以及表2。由圖7以及圖11可以看出，EPA以及DHA是從萃出端E1分離出來，而大部分的其它混合物102是從萃餘端R1分離出來。在切換時間為3分鐘時，包含DHA以及EPA的不飽和脂肪酸的純度可由原本的55.3%提高至87.2%，且具有93.8的回收率。7 to 11 are graphs showing the results of separation and purification of unsaturated fatty acids containing EPA and DHA from 25% fish oil by simulated moving bed chromatography in the experimental example of the present invention. Please refer to FIG. 7 to FIG. 11 and Table 2. As can be seen from Figures 7 and 11, EPA and DHA are separated from the extraction end E1, while most of the other mixture 102 is separated from the raffinate end R1. At a switching time of 3 minutes, the purity of the unsaturated fatty acid containing DHA and EPA was increased from 55.3% to 87.2%, and had a recovery of 93.8.
由上述可知，本實施例的模擬移動床使用包含超臨界二氧化碳與純乙醇的沖滌劑作為移動相，因此可將魚油中的包含DHA以及EPA的不飽和脂肪酸純化分離出來，純度可由原本的55%~66%提升到92%，回收率也可高達84%~96%。 實施例2： EPA 的純化 It can be seen from the above that the simulated moving bed of the present embodiment uses a detergent containing supercritical carbon dioxide and pure ethanol as the mobile phase, so that the unsaturated fatty acid containing DHA and EPA in the fish oil can be purified and purified, and the purity can be 55. %~66% is increased to 92%, and the recovery rate can be as high as 84%~96%. Example 2: Purification of EPA
實驗例2的超臨界流體模擬移動床設備與實施例1（即圖1）所使用的超臨界流體模擬移動床設備相同，因此，相同元件以相同標號表示，且不予贅述。實驗例2的分離純化可以分為兩次的分離步驟（第一模擬移動床層析製程與第二模擬移動床層析製程）。The supercritical fluid simulation moving bed apparatus of Experimental Example 2 is the same as the supercritical fluid simulation moving bed apparatus used in Embodiment 1 (i.e., FIG. 1), and therefore, the same elements are denoted by the same reference numerals and will not be described again. The separation and purification of Experimental Example 2 can be divided into two separation steps (first simulated moving bed chromatography process and second simulated moving bed chromatography process).
詳細來說，在本實驗例的第一模擬移動床層析製程中，是將乙酯化魚油從進料入口F1注入模擬移動床100的第二區段以及第三區段之間，並且使包含EPA以及DHA的不飽脂肪酸隨固定相移動至第一區段與第二區段之間的萃出端E1並使乙酯化魚油中的其它混合物隨移動相移動至第三區段的萃餘端R1。為了達到上述的分離結果，移動相選擇包含超臨界二氧化碳與純乙醇的第一沖滌劑。在本實施例中，以第一沖滌劑的總量計，純乙醇的含量為1 wt%~8 wt%。在一實施例中，以第一沖滌劑的總量計，純乙醇的含量為5 wt%。In detail, in the first simulated moving bed chromatography process of this experimental example, ethyl esterified fish oil is injected from the feed inlet F1 between the second section and the third section of the simulated moving bed 100, and The unsaturated fatty acid comprising EPA and DHA moves with the stationary phase to the extraction end E1 between the first section and the second section and causes the other mixture in the ethyl ester fish oil to move to the third section with the mobile phase The remaining end R1. In order to achieve the above separation results, the mobile phase selects a first detergent comprising supercritical carbon dioxide and pure ethanol. In the present embodiment, the content of pure ethanol is from 1 wt% to 8 wt% based on the total amount of the first detergent. In one embodiment, the pure ethanol is present in an amount of 5 wt% based on the total of the first detergent.
為了進一步將EPA從包含EPA以及DHA的不飽脂肪酸中分離，將上述從萃出端所收集的不飽脂肪酸（含EPA以及DHA）進行第二模擬移動床層析製程。在本實驗例的第二模擬移動床層析製程中，是將第一模擬移動床層析製程中在萃出端E1所收集到的不飽和脂肪酸（含EPA以及DHA）注入模擬移動床100的第二區段與第三區段之間，並使不飽和脂肪酸中的DHA隨固定相移動至第一區段與第二區段之間的萃出端E1，並使不飽和脂肪酸中的EPA隨移動相移動至第三區段的萃餘端R1，以分離EPA以及DHA。為了達到上述的分離結果，移動相選擇包含超臨界二氧化碳與純乙醇的第二沖滌劑。在本實施例中，以第二沖滌劑的總量計，純乙醇的含量為1 wt%~8 wt%。在一實施例中，以第二沖滌劑的總量計，純乙醇的含量為2.5wt%。 分析方法建立 In order to further separate EPA from unsaturated fatty acids containing EPA and DHA, the above-mentioned unsaturated fatty acids (containing EPA and DHA) collected from the extraction end were subjected to a second simulated moving bed chromatography process. In the second simulated moving bed chromatography process of the present experimental example, the unsaturated fatty acid (including EPA and DHA) collected at the extraction end E1 in the first simulated moving bed chromatography process is injected into the simulated moving bed 100. Between the second section and the third section, and moving the DHA in the unsaturated fatty acid to the extraction end E1 between the first section and the second section with the stationary phase, and making the EPA in the unsaturated fatty acid Move to the raffinate end R1 of the third section with the mobile phase to separate the EPA and DHA. In order to achieve the above separation results, the mobile phase selects a second detergent comprising supercritical carbon dioxide and pure ethanol. In the present embodiment, the content of pure ethanol is from 1 wt% to 8 wt% based on the total amount of the second detergent. In one embodiment, the pure ethanol is present in an amount of 2.5% by weight based on the total of the second detergent. Analytical method establishment
分析方法中是使用安捷倫氣相層析質譜儀（GC/MS）（型號7890A/59770B）進行乙酯化魚油的分析，所使用的分析毛細管柱為DB-5MS (30 m L× 250 μm ID，0.25 μm)，並選用1.0毫升/分鐘氦氣作為挾帶氣體。氣相層析質譜儀的升溫條件設定如下：起始120℃並以10℃/分鐘升溫至210℃後持溫10分鐘，再以10℃/分鐘升溫至270℃持溫12分鐘，再以5℃/分鐘升溫至270℃持溫6分鐘，進樣量為1μL，採用分流30:1。 Analytical methods were performed using an Agilent gas chromatography mass spectrometer (GC/MS) (model 7890A/59770B) for the analysis of ethyl ester fish oil using an analytical capillary column of DB-5MS (30 m L × 250 μm ID , 0.25 μm) and 1.0 ml/min of helium is used as the gas. The temperature rising conditions of the gas chromatography mass spectrometer were set as follows: starting at 120 ° C and raising the temperature to 210 ° C at 10 ° C / minute, holding the temperature for 10 minutes, and then raising the temperature to 270 ° C at 10 ° C / minute for 12 minutes, and then 5 The temperature was raised to 270 ° C for 6 minutes at ° C / min, the injection volume was 1 μL, and the split flow was 30:1.
樣品的GC/MS圖譜如圖12所示。圖12為乙酯化魚油的氣相色譜質譜分析圖。在圖12中，內標準品IS採用500毫克/升的十五烷。依據MS資料庫數據比對，在滯留時間為17.5 分鐘處為EPA，而在滯留時間為20.5分鐘處為DHA。從GC/MS圖譜可以清楚判讀出DHA、EPA以及其它混合物102的波鋒位置，藉由此結果做為分析標準。The GC/MS spectrum of the sample is shown in Figure 12. Figure 12 is a gas chromatographic mass spectrometry diagram of ethyl esterified fish oil. In Figure 12, the internal standard IS uses 500 mg/l of pentadecane. According to the MS database data comparison, the EPA was at a residence time of 17.5 minutes, and the DHA was at a residence time of 20.5 minutes. The position of the wave front of DHA, EPA, and other mixtures 102 can be clearly discerned from the GC/MS spectrum, and the results are used as analytical standards.
在本實施例中，製作了EPA以及DHA的檢量線，所得到的響應因子分別為0.308及0.244，上述檢量線搭配十五碳直鏈烷作為內標準品的響應因子（ m），定義如下： (式3) 在式3中， A與 A is 分別為樣品及內標準品在分析圖譜中的面積， C與 C is 為樣品以及內標準品的濃度， V和 V is 為注射料液中樣品與內標準品的體積。 [SF-SMB 分離試驗 ] [ 包含 EPA 與 DHA 的不飽和脂肪酸、 EPA 與 DHA 的純度以及回收率計算 ] In this example, the calibration curves of EPA and DHA were prepared, and the response factors were 0.308 and 0.244, respectively. The calibration curve was matched with fifteen-carbon linear alkane as the response factor ( m ) of the internal standard. as follows: (Formula 3) In Formula 3, A and A is respectively sample and the standards in the analysis pattern of the area, C, and C is the concentration, V and V samples as well as the standard product is an injection feed liquid sample With the volume of the internal standard. [SF-SMB separation test] [contain unsaturated fatty acids EPA and DHA, EPA and DHA recovery and purity calculation]
在第一模擬移動床層析製程的分離實驗主要是先移除圖12中滯留時間短於15分鐘的其它混合物102。由於缺少其它混合物102的標準品，為方便評估分離的成效，因此分離後產物的純度與回收率僅以波鋒面積加以計算。首先，將進料溶液輸入SF-SMB系統以分離成兩種不同組成的樣品。從萃出端得到的樣品按照體積比為9:1的比例與已知濃度的內標物（500毫克/分鐘）均勻混和後，以GC/MS分析得圖譜中EPA與DHA的面積分率，由分析所得圖譜面積計算EPA與DHA兩者的純度及回收率，其計算公式如下式4與式5所示。在本實施例中，EPA與DHA的含量可定義為純度。 (式5) 在式1中，P代表純度，A EPA與A DHA分別為GC/MS圖譜中EPA與DHA的峰面積，SA i表示圖譜中所有的峰面積總和。 The separation experiments in the first simulated moving bed chromatography process primarily removed the other mixture 102 of Figure 12 with a residence time of less than 15 minutes. Due to the lack of standards for other mixtures 102, in order to facilitate the evaluation of the effectiveness of the separation, the purity and recovery of the isolated product are calculated only by the wave front area. First, the feed solution was fed to the SF-SMB system to separate into two samples of different compositions. The sample obtained from the extraction end was uniformly mixed with a known concentration of the internal standard (500 mg/min) in a ratio of 9:1 by volume, and the area fraction of EPA and DHA in the spectrum was analyzed by GC/MS. The purity and recovery of both EPA and DHA were calculated from the analyzed map area, and the calculation formulas are as shown in the following formulas 4 and 5. In this embodiment, the content of EPA and DHA can be defined as purity. (Formula 5) In Formula 1, P represents purity, A EPA and A DHA are the peak areas of EPA and DHA in the GC/MS spectrum, respectively, and SA i represents the sum of all peak areas in the spectrum.
回收率則依以下式6來估算。 (式6) 在式6中，Y代表回收率，A EPA與A DHA分別為GC/MS圖譜中EPA與DHA的峰面積，上標E與R分別代表萃出端以及萃餘端，而Q為乙醇的體積流速。 The recovery rate is estimated by the following formula 6. (Formula 6) In Equation 6, Y represents the recovery rate, A EPA and A DHA are the peak areas of EPA and DHA in the GC/MS spectrum, respectively, and the superscripts E and R represent the extraction end and the raffinate end, respectively, and Q It is the volumetric flow rate of ethanol.
第二模擬移動床層析製程的主要目的在分離DHA與EPA，同時EPA與DHA也容易製作出檢量線，因此針對第二模擬移動床層析製程的實驗，本實施例定義兩個出口端的純度與回收率如以下式7與式8所示。 (式7) 在式1中，P代表純度，C EPA與C DHA分別為GC/MS圖譜中回歸計算所得到的EPA與DHA的濃度，上標E與R分別代表萃出端以及萃餘端。 The main purpose of the second simulated moving bed chromatography process is to separate DHA from EPA, and EPA and DHA are also easy to make a calibration curve. Therefore, for the experiment of the second simulated moving bed chromatography process, this embodiment defines two outlet ends. The purity and recovery are as shown in the following formulas 7 and 8. (Formula 7) In Formula 1, P represents purity, and C EPA and C DHA are the concentrations of EPA and DHA obtained by regression calculation in the GC/MS spectrum, respectively. Superscripts E and R represent the extraction end and the raffinate end, respectively. .
回收率則依以下式8來估算。 (式8) 在式8中，Y代表回收率，C EPA與C DHA分別為GC/MS圖譜中回歸計算所得到的EPA與DHA的濃度，上標E與R分別代表萃出端以及萃餘端，而Q為乙醇的體積流速。 實驗例 3 [SF-SMB 的操作條件 ] The recovery rate is estimated by the following formula 8. (Formula 8) In Equation 8, Y represents the recovery rate, C EPA and C DHA are the concentrations of EPA and DHA obtained by regression calculation in the GC/MS spectrum, respectively, and the superscripts E and R represent the extraction end and the raffinate, respectively. Terminal, and Q is the volumetric flow rate of ethanol. Experimental Example 3 [ Operation conditions of SF-SMB ]
在實驗例3中，將乙酯化魚油原料先配製成9.823 克/升的乙醇溶液。接著，使用圖1所示的超臨界流體模擬移動床設備來進行模擬移動床層析法。填充管柱為80 mm的DAC管柱，所採用的填料（固定相）為無規二氧化矽（Zeoprep60，40 μm~60 μm，Zeochem），填充高度為230 mm。而移動相為包含超臨界二氧化碳與5wt%純乙醇的沖滌劑。分離的條件為：溫度固定為50℃，萃餘端出口壓力為121 bar，而沖滌劑入口壓力130 bar。各入口與出口端的二氧化碳流速設定如下：沖滌端入口為26.5 公斤/小時；進料入口為1.51 公斤/小時；萃出端為11.78 公斤/小時；萃餘端為16.32 公斤/小時（經質量守恆計算）。入料口的純乙醇流速設定如下：沖滌端入口為29.39 毫升/分鐘；進料入口為1.65 毫升/分鐘；萃出端為13.1毫升/分鐘；萃餘端的乙醇流速為17.94 毫升/分鐘（經質量守恆計算）。此外，在實驗例3中，在固定各出入口的流速條件下，改變SF-SMB設備上閥門的切換時間（3分鐘30秒、4分鐘15秒與5分鐘），然後觀察二個出料口所收集樣品的組成隨切換時間的變化。In Experimental Example 3, the ethyl esterified fish oil raw material was first formulated into a 9.823 g/liter ethanol solution. Next, simulated moving bed chromatography was carried out using the supercritical fluid simulated moving bed apparatus shown in FIG. The packed column is an 80 mm DAC column with a packing (stationary phase) of random cerium oxide (Zeoprep 60, 40 μm~60 μm, Zeochem) with a fill height of 230 mm. The mobile phase is a detergent containing supercritical carbon dioxide and 5 wt% pure ethanol. The conditions for separation were: a temperature of 50 ° C, a raffinate outlet pressure of 121 bar, and a detergent inlet pressure of 130 bar. The carbon dioxide flow rate at each inlet and outlet is set as follows: 26.5 kg/h for the flush inlet; 1.51 kg/hr for the feed inlet; 11.78 kg/hr for the extraction end; 16.32 kg/hr for the raffinate end (conservation of mass) Calculation). The pure ethanol flow rate at the feed port was set as follows: the flush inlet end was 29.39 ml/min; the feed inlet was 1.65 ml/min; the extraction end was 13.1 ml/min; and the raffinate end ethanol flow rate was 17.94 ml/min (via Mass conservation calculation). Further, in Experimental Example 3, the switching time of the valve on the SF-SMB device was changed (3 minutes, 30 seconds, 4 minutes, 15 seconds, and 5 minutes) under the condition of fixing the flow rate of each inlet and outlet, and then the two discharge ports were observed. The composition of the collected samples varies with the switching time.
切換時間3分鐘30秒以及5分鐘的分離結果分別為萃出端以及萃餘端的泛流。圖13為模擬移動床的切換時間為4分鐘15秒的結果分析圖，且依據式5以及式6所計算純度與回收率的結果如表3所示。由圖13的結果可以看出，在切換時間為4分鐘15秒時，可以從魚油中分離純化出包含DHA以及EPA的不飽和脂肪酸，而且可推測DHA為為魚油中最強滯留成分，其次為EPA。 實驗例 4 [SF-SMB 的操作條件 ] The separation time of the switching time of 3 minutes and 30 seconds and 5 minutes was the flooding of the extraction end and the raffinate end, respectively. Fig. 13 is a graph showing the results of the simulation of the switching time of the moving bed of 4 minutes and 15 seconds, and the results of the purity and the recovery calculated according to the formulas 5 and 6 are shown in Table 3. It can be seen from the results of Fig. 13 that when the switching time is 4 minutes and 15 seconds, the unsaturated fatty acid containing DHA and EPA can be separated and purified from the fish oil, and it can be presumed that DHA is the strongest retention component in fish oil, followed by EPA. . Experimental Example 4 [ Operation conditions of SF-SMB ]
在實驗例4中，將乙酯化魚油原料先配製成9.823 克/升的乙醇溶液。接著，使用圖1所示的超臨界流體模擬移動床設備來進行模擬移動床層析法。填充管柱為80 mm的DAC管柱，所採用的填料（固定相）為無規二氧化矽（Zeoprep60，40 μm~60 μm，Zeochem），填充高度為230 mm。而移動相為包含超臨界二氧化碳與5wt%純乙醇的沖滌劑。分離的條件為：溫度固定為50℃，萃餘端出口壓力為121 bar，而沖滌劑入口壓力130 bar。各入口與出口端的二氧化碳流速設定如下：沖滌端入口為26.5 公斤/小時；進料入口為1.05 公斤/小時；萃出端為11.78 公斤/小時；萃餘端為15.77 公斤/小時（經質量守恆計算）。入料口的純乙醇流速設定如下：沖滌端入口為29.39 毫升/分鐘；進料入口為1.12 毫升/分鐘；萃出端為13.1毫升/分鐘；萃餘端的乙醇流速為17.4 毫升/分鐘（經質量守恆計算）。此外，在實驗例4中，模擬移動床的切換時間為4分鐘。採用上述條件進行模擬移動床層析法所得到的結果分析如圖14所示，且依據式5以及式6所計算純度與回收率的結果如表3所示。In Experimental Example 4, the ethyl esterified fish oil raw material was first formulated into a 9.823 g/liter ethanol solution. Next, simulated moving bed chromatography was carried out using the supercritical fluid simulated moving bed apparatus shown in FIG. The packed column is an 80 mm DAC column with a packing (stationary phase) of random cerium oxide (Zeoprep 60, 40 μm~60 μm, Zeochem) with a fill height of 230 mm. The mobile phase is a detergent containing supercritical carbon dioxide and 5 wt% pure ethanol. The conditions for separation were: a temperature of 50 ° C, a raffinate outlet pressure of 121 bar, and a detergent inlet pressure of 130 bar. The carbon dioxide flow rate at each inlet and outlet is set as follows: 26.5 kg/h for the flush inlet; 1.05 kg/hr for the feed inlet; 11.78 kg/hr for the extraction end; 15.77 kg/hr for the raffinate end (conservation of mass) Calculation). The pure ethanol flow rate at the feed port was set as follows: the flush inlet end was 29.39 ml/min; the feed inlet was 1.12 ml/min; the extraction end was 13.1 ml/min; and the raffinate end ethanol flow rate was 17.4 ml/min (via Mass conservation calculation). Further, in Experimental Example 4, the switching time of the simulated moving bed was 4 minutes. The results obtained by the simulated moving bed chromatography using the above conditions are shown in Fig. 14, and the results of the purity and recovery calculated according to the formulas 5 and 6 are shown in Table 3.
表3 <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> 純度 (%) </td><td> 回收率 (%) </td></tr><tr><td> 實驗例3 切換時間 4分鐘15秒 </td><td> 86.8 </td><td> 27.6 </td></tr><tr><td> 實驗例4 切換時間 4分鐘 </td><td> 87.8 </td><td> 91.6 </td></tr></TBODY></TABLE>table 3 <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> Purity (%) </td><td> Recovery rate ( %) </td></tr><tr><td> Experimental Example 3 Switching time 4 minutes 15 seconds</td><td> 86.8 </td><td> 27.6 </td></tr>< Tr><td> Experimental Example 4 Switching time 4 minutes</td><td> 87.8 </td><td> 91.6 </td></tr></TBODY></TABLE>
圖14為模擬移動床的切換時間為4分鐘的結果分析圖。請參照圖13、圖14以及表3，實驗例4 的操作條件由於降低進料流速以及縮短切換時間（相對於實驗例3的操作條件），可以成功地分離從魚油中分離純化出包含DHA以及EPA的不飽和脂肪酸，其純度及回收率可分別達87.8%以及91.6%。 實驗例 5 [SF-SMB 的操作條件 ] Fig. 14 is a graph showing the result of simulating the switching time of the moving bed of 4 minutes. Referring to FIG. 13 , FIG. 14 and Table 3, the operating conditions of Experimental Example 4 can be successfully separated and purified from fish oil by using DHA as well as reducing the feed flow rate and shortening the switching time (relative to the operating conditions of Experimental Example 3). The purity and recovery rate of EPA unsaturated fatty acids can reach 87.8% and 91.6%, respectively. Experimental Example 5 [ Operation conditions of SF-SMB ]
在實驗例5中，將實驗例4中在萃出端E1所收集的包含DHA以及EPA的不飽和脂肪酸作為進行第二模擬移動床層析製程的進料，且將上述進料調整為20.23 克/升的乙醇溶液。此外，在實驗例5中，使用與實驗例3相同的超臨界流體模擬移動床設備來進行模擬移動床層析法。填充管柱為80 mm的DAC管柱，所採用的填料（固定相）為無規二氧化矽（Zeoprep60，40 μm~60 μm，Zeochem），填充高度為230 mm。而移動相為包含超臨界二氧化碳與2.5wt%純乙醇的沖滌劑。分離的條件為：溫度固定為50℃，萃餘端出口壓力為121 bar，而沖滌劑入口壓力130 bar。各入口與出口端的二氧化碳流速設定如下：沖滌端入口為26.5 公斤/小時；進料入口為0.3 公斤/小時；萃出端為11.78 公斤/小時；萃餘端為15.02 公斤/小時（經質量守恆計算）。入料口的純乙醇流速設定如下：沖滌端入口為14.7 毫升/分鐘；進料入口為0.165 毫升/分鐘；萃出端為6.55毫升/分鐘；萃餘端的乙醇流速為8.315 毫升/分鐘（經質量守恆計算）。此外，在實驗例5中，在固定各出入口的流速條件下，改變SF-SMB設備上閥門的切換時間（5分鐘15秒、5分鐘30秒與5分鐘40秒），然後觀察二個出料口所收集樣品的組成隨切換時間的變化。採用上述條件進行模擬移動床層析法所得到的結果分析如圖15至圖17所示，且依據式7以及式8所計算純度與回收率的結果如表4所示。In Experimental Example 5, the unsaturated fatty acid containing DHA and EPA collected at the extraction end E1 in Experimental Example 4 was used as a feed for the second simulated moving bed chromatography process, and the above feed was adjusted to 20.23 g. / liter of ethanol solution. Further, in Experimental Example 5, simulated moving bed chromatography was carried out using the same supercritical fluid simulated moving bed apparatus as Experimental Example 3. The packed column is an 80 mm DAC column with a packing (stationary phase) of random cerium oxide (Zeoprep 60, 40 μm~60 μm, Zeochem) with a fill height of 230 mm. The mobile phase is a detergent containing supercritical carbon dioxide and 2.5 wt% pure ethanol. The conditions for separation were: a temperature of 50 ° C, a raffinate outlet pressure of 121 bar, and a detergent inlet pressure of 130 bar. The carbon dioxide flow rate at each inlet and outlet is set as follows: 26.5 kg/h for the flush inlet; 0.3 kg/hr for the feed inlet; 11.78 kg/hr for the extraction end; 15.02 kg/hr for the raffinate end (conservation of mass) Calculation). The pure ethanol flow rate at the feed port was set as follows: 14.7 ml/min for the flush inlet; 0.165 ml/min for the feed inlet; 6.55 ml/min for the extraction end; and 8.315 ml/min for the raffinate end (via Mass conservation calculation). Further, in Experimental Example 5, the switching time of the valve on the SF-SMB device was changed (5 minutes, 15 seconds, 5 minutes, 30 seconds, and 5 minutes and 40 seconds) under the condition of fixing the flow rate of each inlet and outlet, and then two discharges were observed. The composition of the sample collected by the mouth varies with the switching time. The results obtained by the simulated moving bed chromatography using the above conditions were analyzed as shown in Figs. 15 to 17, and the results of the purity and recovery calculated according to the formulas 7 and 8 are shown in Table 4.
表4 <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> 切換時間 </td><td> 濃度(m克/升) </td><td> 純度(%) </td><td> 回收率(%) </td></tr><tr><td> 萃出液 </td><td> 萃餘液 </td><td> EPA </td><td> DHA </td><td> EPA </td><td> DHA </td></tr><tr><td> EPA </td><td> DHA </td><td> EPA </td><td> DHA </td></tr><tr><td> 進料 </td><td> 11445.8 </td><td> 3144.0 </td><td> － </td><td> － </td><td> 78.5 </td><td> － </td><td> － </td><td> － </td></tr><tr><td> 5分鐘15秒 </td><td> 82.1 </td><td> 50.1 </td><td> 20.7 </td><td> 0.0 </td><td> 100.0 </td><td> 62.1 </td><td> 24.4 </td><td> 100.0 </td></tr><tr><td> 5分鐘30秒 </td><td> 71.5 </td><td> 50.0 </td><td> 192.8 </td><td> 4.7 </td><td> 97.6 </td><td> 58.8 </td><td> 77.5 </td><td> 89.3 </td></tr><tr><td> 5分鐘40秒 </td><td> 72.2 </td><td> 67.0 </td><td> 132.1 </td><td> 3.8 </td><td> 97.2 </td><td> 51.8 </td><td> 70.0 </td><td> 93.2 </td></tr></TBODY></TABLE>Table 4 <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> Switching time</td><td> Concentration (mg/L) </td>< Td> purity (%) </td><td> recovery rate (%) </td></tr><tr><td> extract solution</td><td> raffinate solution</td>< Td> EPA </td><td> DHA </td><td> EPA </td><td> DHA </td></tr><tr><td> EPA </td><td> DHA </td><td> EPA </td><td> DHA </td></tr><tr><td> Feed </td><td> 11445.8 </td><td> 3144.0 </ Td><td> - </td><td> - </td><td> 78.5 </td><td> - </td><td> - </td><td> - </td> </tr><tr><td> 5 minutes 15 seconds</td><td> 82.1 </td><td> 50.1 </td><td> 20.7 </td><td> 0.0 </td> <td> 100.0 </td><td> 62.1 </td><td> 24.4 </td><td> 100.0 </td></tr><tr><td> 5 minutes 30 seconds</td> <td> 71.5 </td><td> 50.0 </td><td> 192.8 </td><td> 4.7 </td><td> 97.6 </td><td> 58.8 </td><td > 77.5 </td><td> 89.3 </td></tr><tr><td> 5 minutes and 40 seconds</td><td> 72.2 </td><td> 67.0 </td><td > 132.1 </td><td> 3.8 </td><td> 97.2 </td><td> 51.8 </td><td> 70.0 </td><td> 93.2 </td></tr> </TBODY></TABLE>
圖15至圖17為本發明實驗例利用模擬移動床層析法從不飽和脂肪酸中分離純化出EPA以及DHA的結果分析圖。請參照圖15至圖17以及表4。在切換時間為5分鐘15秒時，萃餘端可以收集到純度高達100%的EPA。在切換時間為5分鐘30秒時，EPA的純度為97.6%，且回收率也可達77.5%。且在切換時間為5分鐘40秒時，EPA的純度為97.2%，且回收率也可達70%。由上述的內容可知，由於實驗例5的第二模擬移動床層析製程的操作條件採用較低的純乙醇濃度（2.5wt%），因此可純化出高純度的EPA。15 to 17 are graphs showing the results of separation and purification of EPA and DHA from unsaturated fatty acids by simulated moving bed chromatography in an experimental example of the present invention. Please refer to FIG. 15 to FIG. 17 and Table 4. At a switching time of 5 minutes and 15 seconds, the raffinate can collect EPA with a purity of up to 100%. At a switching time of 5 minutes and 30 seconds, the purity of the EPA was 97.6%, and the recovery rate was also 77.5%. And when the switching time is 5 minutes and 40 seconds, the purity of EPA is 97.2%, and the recovery rate can reach 70%. From the above, it can be seen that since the operating conditions of the second simulated moving bed chromatography process of Experimental Example 5 employ a lower pure ethanol concentration (2.5 wt%), high purity EPA can be purified.
綜上所述，本發明的不飽和脂肪酸的純化方法透過應用模擬移動床層析法來從魚油中分離包含EPA以及DHA的不飽和脂肪酸，不僅可有效提升分離效率，更可獲得高純度的包含EPA以及DHA的不飽和脂肪酸。此外，本發明的二十碳五烯酸的純化方法可藉由進行二次模擬移動床層析製程而進一步從魚油中純化出二十碳五烯酸，同樣地，不僅可有效提升分離效率，更可獲得高純度的二十碳五烯酸。In summary, the method for purifying the unsaturated fatty acid of the present invention separates the unsaturated fatty acid containing EPA and DHA from the fish oil by using simulated moving bed chromatography, thereby not only effectively improving the separation efficiency, but also obtaining high purity inclusion. Unsaturated fatty acids from EPA and DHA. In addition, the purification method of eicosapentaenoic acid of the present invention can further purify eicosapentaenoic acid from fish oil by performing a secondary simulated moving bed chromatography process, and similarly, not only can the separation efficiency be effectively improved, More high-purity eicosapentaenoic acid is obtained.
雖然本發明已以實施例揭露如上，然其並非用以限定本發明，任何所屬技術領域中具有通常知識者，在不脫離本發明的精神和範圍內，當可作些許的更動與潤飾，故本發明的保護範圍當視後附的申請專利範圍所界定者為準。Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and any one of ordinary skill in the art can make some changes and refinements without departing from the spirit and scope of the present invention. The scope of the invention is defined by the scope of the appended claims.
100‧‧‧模擬移動床100‧‧‧simulated moving bed
102‧‧‧其它混合物102‧‧‧Other mixtures
110‧‧‧二氧化碳供應源110‧‧‧source of carbon dioxide
115‧‧‧二氧化碳液泵115‧‧‧Carbon dioxide pump
120‧‧‧高壓緩衝槽120‧‧‧High pressure buffer tank
122‧‧‧前端壓力調壓閥122‧‧‧ front pressure regulator
123‧‧‧後端壓力調壓閥123‧‧‧Back pressure regulator
125a、125b‧‧‧高效能液相層析液泵125a, 125b‧‧‧ high performance liquid chromatography pump
130‧‧‧混合器130‧‧‧mixer
145a、145b、155‧‧‧分離槽145a, 145b, 155‧‧ separate tank
160‧‧‧工作儲槽160‧‧‧Work storage tank
C1、C2、C3、C4、C5、C6、C7、C8‧‧‧管柱C1, C2, C3, C4, C5, C6, C7, C8‧‧‧ column
D1‧‧‧沖滌端入口D1‧‧‧Driving end entrance
D2、F2‧‧‧輸入口D2, F2‧‧‧ input port
E1‧‧‧萃出端E1‧‧‧ extraction end
F1‧‧‧進料入口F1‧‧‧ Feed inlet
IS‧‧‧內標準品IS‧‧‧standards
R1‧‧‧萃餘端R1‧‧‧
圖1是依照本發明實施例的一種超臨界流體模擬移動床設備的管線流程圖。 圖2為10%乙酯化魚油的氣相色譜質譜分析圖。 圖3為25%乙酯化魚油的氣相色譜質譜分析圖。 圖4至圖6為本發明實驗例利用模擬移動床層析法從10%魚油中分離純化出包含EPA以及DHA的不飽和脂肪酸的結果分析圖。 圖7至圖11為本發明實驗例利用模擬移動床層析法從25%魚油中分離純化出包含EPA以及DHA的不飽和脂肪酸的結果分析圖。 圖12為乙酯化魚油的氣相色譜質譜分析圖。 圖13為模擬移動床的切換時間為4分鐘15秒的結果分析圖。 圖14為模擬移動床的切換時間為4分鐘的結果分析圖。 圖15至圖17為本發明實驗例利用模擬移動床層析法從不飽和脂肪酸中分離純化出EPA以及DHA的結果分析圖。1 is a pipeline flow diagram of a supercritical fluid simulated moving bed apparatus in accordance with an embodiment of the present invention. Figure 2 is a gas chromatographic mass spectrogram of 10% ethyl ester fish oil. Figure 3 is a gas chromatographic mass spectrogram of 25% ethyl esterified fish oil. 4 to 6 are graphs showing the results of separation and purification of unsaturated fatty acids containing EPA and DHA from 10% fish oil by simulated moving bed chromatography in the experimental example of the present invention. 7 to 11 are graphs showing the results of separation and purification of unsaturated fatty acids containing EPA and DHA from 25% fish oil by simulated moving bed chromatography in the experimental example of the present invention. Figure 12 is a gas chromatographic mass spectrometry diagram of ethyl esterified fish oil. Fig. 13 is a graph showing the result of the simulation of the switching time of the moving bed of 4 minutes and 15 seconds. Fig. 14 is a graph showing the result of simulating the switching time of the moving bed of 4 minutes. 15 to 17 are graphs showing the results of separation and purification of EPA and DHA from unsaturated fatty acids by simulated moving bed chromatography in an experimental example of the present invention.
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