WO2024055337A1 - 一种香榧籽油中多种高纯度不饱和脂肪酸的分离方法 - Google Patents

一种香榧籽油中多种高纯度不饱和脂肪酸的分离方法 Download PDF

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WO2024055337A1
WO2024055337A1 PCT/CN2022/119492 CN2022119492W WO2024055337A1 WO 2024055337 A1 WO2024055337 A1 WO 2024055337A1 CN 2022119492 W CN2022119492 W CN 2022119492W WO 2024055337 A1 WO2024055337 A1 WO 2024055337A1
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fatty acids
seed oil
unsaturated fatty
acid
phase
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高锦明
杨志
李岚洁
高昱昕
杨景晖
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西北农林科技大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/09—Preparation of carboxylic acids or their salts, halides or anhydrides from carboxylic acid esters or lactones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/48—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/02—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms with only carbon-to-carbon double bonds as unsaturation
    • C07C57/03—Monocarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/02—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms with only carbon-to-carbon double bonds as unsaturation
    • C07C57/03—Monocarboxylic acids
    • C07C57/12—Straight chain carboxylic acids containing eighteen carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils

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  • the present invention relates to a method for separating nutrients in torreya seed oil, and specifically relates to a method for separating multiple high-purity unsaturated fatty acids from torreya seed oil.
  • Torreya grandis also known as Torreya grandis, also known as Torreya grandis, Torreya grandis, etc.
  • Torreya grandis is an evergreen tree of the order Taxaceae, Taxaceae, and is a tree species native to China. It is a rare economic tree species in the world. , its fruit is wrapped by a hard peel, the size is like a date, the pit is like an olive, the ends are pointed, and it is oval. When mature, the shell is yellow-brown or purple-brown, and the seeds are yellow-white, rich in oil and a unique aroma. Torreya seeds are not only rich in nutrients, but also have many functions and effects. They are recognized as natural beauty foods and medicinal materials. Regular consumption can moisturize the skin and delay aging.
  • torreya seeds can eliminate malnutrition, It has the functions of moistening the lungs, smoothing the intestines, resolving phlegm and relieving cough, and is suitable for various symptoms of constipation, hernia, hemorrhoids, indigestion, food accumulation and expectoration. Modern research shows that torreya seed extract also has physiological functions such as antioxidant, anti-acute inflammation, and lowering blood lipids.
  • the unsaturated fatty acid content in torreya seed oil can reach up to about 90%.
  • the torreya seed oil mainly contains linoleic acid (39.85% ⁇ 46.15%), oleic acid (22.68% ⁇ 35.10%), and golden acid (9.13% ⁇ 12.96%) , palmitic acid (7.34% ⁇ 8.22%), stearic acid (2.64% ⁇ 3.03%) and other 9 kinds of fatty acids.
  • corticoic acid has anti-inflammatory, anti-viral, immunity-improving effects, and has various physiological activities such as regulating blood lipids, preventing atherosclerosis, and promoting metabolism.
  • CN109796328A uses regioselective lipase TLIM or RMIM to moderately catalyze the transesterification reaction of torreya seed oil and ethanol to achieve the initial enrichment of golden pinic acid, and then urea inclusion removes saturated fatty acids and low saturation unsaturated fatty acids to further purify golden pinnic acid.
  • TLIM regioselective lipase
  • RMIM regioselective lipase
  • the lipase used is expensive and the production cost is high; in addition, the purity of the obtained product is above 90%, and the purity is still difficult to meet the requirements
  • the purity has been improved to a certain extent, but the use of lipase Increase production costs; in addition, the urea inclusion enrichment product is separated by silver ion chromatography. Although the purity of the product can be as high as 99%, the cost is also high.
  • CN112679343A first uses sodium ethoxide to convert the golden acid in torreya seed oil into ethyl golden acid, and then uses silver ions as the coupling agent to separate and purify it by counter-current chromatography.
  • the prices of sodium ethoxide and silver ion salts used in this method are both It is relatively high and the production cost is huge; in addition, the purity of the product is only 20-85%, which is relatively low.
  • the present invention provides a method for separating multiple high-purity unsaturated fatty acids in torreya seed oil.
  • the separation method of the present invention includes the following steps:
  • the sample solution is separated by counter-current chromatography to obtain a variety of unsaturated fatty acids.
  • the stationary phase of counter-current chromatography separation is the upper phase, and the mobile phase is the lower phase; the pumping speed of the mobile phase is 1-10 mL/min.
  • the detection wavelength is 190-230nm, the column temperature is 20-40°C; the countercurrent chromatography instrument speed is 500-1500rpm.
  • the pumping speed of the mobile phase is 4mL/min
  • the detection wavelength is 210nm
  • the column temperature is 30°C
  • the instrument rotation speed is 1000rpm
  • the elution time is 115min-220min.
  • isolates with elution times of 115-158min, 165-179min, 185-195min, and 205-220min were collected to obtain linoleic acid, auronic acid, oleic acid and eicosadienoic acid.
  • the organic solvent is heptane, pentane, hexane, petroleum ether or ethyl acetate.
  • step (2) the heptane, absolute ethanol and water are mixed in a volume ratio of 10:9:1.
  • step (3) the mixture of heptane, absolute ethanol and water is replaced with a mixture of heptane, methanol and water, a mixture of hexane, methanol and water or hexane and absolute ethanol. and water mixture.
  • the present invention uses counter-current chromatography to separate the hydrolyzate of torreya seed oil, and can simultaneously obtain high-purity four unsaturated fatty acid compounds, namely, golden acid, oleic acid, linoleic acid and eicosadienoic acid.
  • the specific plan uses an alkaline solution to hydrolyze the sample. After acidification, fatty acid products such as auric acid are obtained directly through extraction.
  • a single counter-current chromatographic separation the separation and purification of multiple unsaturated fatty acid components in torreya seed oil is achieved, effectively shortening the separation time. Preparation time; and this method is not only low in cost, but also simple and reliable in operation, which provides technical support for the development and utilization of unsaturated fatty acids in torreya seed oil and has good application value.
  • Figure 1 is a countercurrent chromatography separation spectrum of separation in Example 1;
  • Figure 2 is a HPLC analysis chart of the isolate obtained in Example 1;
  • Figure 3 is a countercurrent chromatographic separation diagram of Example 2.
  • Figure 4 is an HPLC analysis chart of the segmented product of Example 2.
  • the countercurrent chromatograph used in the following implementation is the OptiChrome-300PLUS high-speed countercurrent chromatograph produced by Jiangyin Countercurrent Technology Co., Ltd.
  • the torreya seed oil and reagents used were all commercially available products.
  • the isolates with elution time of 115-220 minutes were collected and subjected to HPLC detection and NMR detection.
  • the conditions for HPLC analysis are: the chromatographic column is a YMC-C18 column (id 4.6 ⁇ 150mm, 5 ⁇ m); gradient washing is performed with methanol (A) and trifluoroacetic acid (0.03%, v/v) solution (B) as the mobile phase.
  • the gradient elution conditions are 0 to 15 min, 80%-100% A, 15 to 25 min, 100% A; the column temperature is 30°C; the flow rate is 0.8mL/min; the injection volume is 5 ⁇ L; the detection wavelength is 210nm.
  • Figure 2 is the HPLC analysis spectrum of the free fatty acids separated in Example 1.
  • Example 1 The separation results of Example 1 are shown in Figure 1. The separation was completed within 4 hours. After HPLC analysis, the products were combined to obtain high-purity linoleic acid, golden acid, oleic acid, and eicosadienoic acid products.
  • the masses of linoleic acid, golden pinic acid, oleic acid, and eicosadienoic acid are: 39 mg, 18 mg, 13 mg, and 7 mg respectively; and the area normalization method is used to calculate the sample purity.
  • the separated linoleic acid, golden pinnic acid, The purity of oleic acid and eicosadienoic acid are: 98%, 96%, 94%, and 96% respectively.
  • Example 1 The difference between this embodiment and Example 1 is that the heptane, absolute ethanol and water solvent systems are replaced with n-heptane, ethyl acetate, methanol and water solvent systems with a volume ratio of 5:5:9:1; 70- The products were separated at 80 min and 85-95 min.
  • Figure 3 is a countercurrent chromatographic separation diagram of Example 2
  • Figure 4 is an HPLC analysis diagram of the countercurrent segmented product of Example 2. It can be seen from the figure that the four fatty acids are not effectively separated, and the separated product is a mixture of several fatty acids.

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Abstract

本发明公开了一种香榧籽油中多种高纯度不饱和脂肪酸的分离方法，所述分离方法解决了现有技术分离物单一及成本高的问题。所公开的方案采用碱溶液对样品进行水解，酸化后直接通过萃取获得粗提物，通过一次逆流色谱分离便实现了香榧籽油中金松酸、油酸、亚油酸以及二十碳二烯酸多种不饱和脂肪酸成分的分离纯化，有效地缩短了分离制备的时间；并且该方法不仅成本低，而且操作简单可靠，为香榧籽油中不饱和脂肪酸的开发利用提供了技术支撑，具有较好的应用价值。

Description

一种香榧籽油中多种高纯度不饱和脂肪酸的分离方法 技术领域
本发明涉及香榧籽油中营养物的分离方法，具体涉及一种香榧籽油中多种高纯度不饱和脂肪酸的分离方法。
背景技术
香榧(Torreya grandis)，又称赤果、玉山果、玉榧、野极子等，为红豆杉目、红豆杉科、榧树属常绿乔木，中国原产树种，是世界上稀有的经济树种，其果实外有坚硬的果皮包裹，大小如枣，核如橄榄，两头尖，呈椭圆形，成熟后果壳为黄褐色或紫褐色，种实为黄白色，富有油脂和特有的一种香气。香榧子不但营养丰富，它的功效和作用也非常多，是公认的天然的美颜食品和药材，经常食用有润泽肌肤、延缓衰老的作用；其次传统中医药学认为，香榧子具有消除疳积、润肺滑肠、化痰止咳之功能，并且适用于多种便秘、疝气、痔疮、消化不良、食积、咳痰症状。现代研究表明，香榧子提取物还具有抗氧化、抗急性炎性以及降血脂等的生理功能。
香榧籽油中不饱和脂肪酸含量最高可以达到90％左右，香榧籽油主要含有亚油酸(39.85％～46.15％)、油酸(22.68％～35.10％)、金松酸(9.13％～12.96％)、棕榈酸(7.34％～8.22％)、硬脂酸(2.64％～3.03％)等9种脂肪酸。其中金松酸具有抗炎、抗病毒、提高免疫力的效果，具有调节血脂、预防动脉粥样硬化、促进新陈代谢等多种生理活性。
现有针对香榧籽油中不饱和脂肪酸的纯化方法主要有脂肪酸选择性醇解结合尿素包合法、逆流色谱耦合银离子络合法等，并且相关的方法主要集 中在金松酸的分离纯化方面，且成本高。例如：
CN109796328A用区域选择性脂肪酶TLIM或RMIM适度催化香榧籽油、乙醇酯交换反应，实现金松酸初步富集，然后尿素包合除去饱和脂肪酸与低饱和度不饱和脂肪酸后进一步纯化金松酸，该方法所用脂肪酶价格昂贵，生产成本高；另外，获得的产品的纯度在90％以上，纯度依然难以满足医药等行业的要求。
孟祥河等(参见，孟祥河,杨奇波,肖丹,夏朝盛,樊律廷,宋丽丽,吴家胜.香榧籽油金松酸的分离及其1,3-甘油二酯的制备研究[J].中国粮油学报,2020,35(07):72-78)采用酯交换反应结合尿素包合法，将香榧籽油中金松酸的含量提升到了60-73％，但产品的纯度较低。
孟祥河等(参见，Xianghe Meng,Dan Xiao,Qin Ye,Xiaohua Nie,Jiasheng Wu,Lili Song.Positional distribution ofΔ5-olefinic acids in triacylglycerols from Torreya grandis seed oil:Isolation and purification ofsciadonic acid[J].Industrial Crops&Products,2020,143)又利用脂肪酶选择性醇解和尿素包合两步富集过程，将香榧籽油中金松酸的含量由9.95％提高到了80.14％，纯度得到了一定的提升，但脂肪酶的使用增加生产成本；另外，借助银离子色谱法对尿素包合富集产物进行分离，产品的纯度虽然可以高达99％，但成本同样高昂。
Hammann等(参见，Simon Hammann,Markus
Figure PCTCN2022119492-appb-000001
Carolin Schmidt,Walter Vetter.Isolation of twoΔ5polymethylene interrupted fatty acids from Podocarpus falcatus by countercurrent chromatography[J].Journal of ChromatographyA,2015,1394)通过三步逆流色谱分离得到纯度为99％的金松酸，但使用的溶剂毒性较高，同时产品的回收率较低。
CN112679343A首先利用乙醇钠将香榧籽油中的金松酸转化成金松酸 乙酯，然后选用银离子作为耦合剂，采用逆流色谱对其进行分离纯化,该方法所用的乙醇钠和银离子盐的价格都较高，生产成本巨大；另外，产品的纯度只有20-85％，纯度较低。
发明内容
针对现有技术的缺陷或不足，本发明提供了一种香榧籽油中多种高纯度不饱和脂肪酸的分离方法。
为此，本发明的分离方法包括以下步骤：
(1)将香榧籽油与碱性水溶液在40-80℃条件下回流反应，反应完成后用1M盐酸或冰乙酸调整反应物pH值为3-5后，用有机溶剂萃取pH值为3-5的所述反应物，所得萃取液经浓缩得浓缩物；所述碱性水溶液为KOH水溶液或NaOH水溶液；
(2)所述浓缩物用上相和下相溶解后得样品溶液；庚烷、无水乙醇和水混合后静置分离上层液体和下层液体，其中上层液体为所述上相、下层液体为所述下相；
(3)所述样品溶液用逆流色谱分离得多种不饱和脂肪酸，逆流色谱分离的固定相为所述上相，流动相为所述下相；流动相的泵送速度为1-10mL/min，检测波长为190-230nm，柱温为20-40℃；逆流色谱仪器转速为500-1500rpm。
进一步，流动相的所述泵送速度为4mL/min，所述检测波长为210nm，所述柱温为30℃，所述仪器转速为1000rpm，收集洗脱时间为115min-220min时间段的分离物得到多种不饱和脂肪酸。
进一步，分别收集洗脱时间为115-158min、165-179min、185-195min、 205-220min时间段的分离物得到亚油酸、金松酸、油酸和二十碳二烯酸。
可选地，在步骤(1)中，所述有机溶剂选用庚烷、戊烷、己烷、石油醚或者乙酸乙酯。
可选地，在步骤(2)中，所述庚烷、无水乙醇和水按体积比10:9:1混合。
可选地，在步骤(3)中，所述庚烷、无水乙醇和水混合液替换为庚烷、甲醇和水的混合液，己烷、甲醇和水混合液或者己烷、无水乙醇和水混合液。
本发明利用逆流色谱对香榧籽油水解产物进行分离，可以同时获得高纯度的金松酸、油酸、亚油酸以及二十碳二烯酸4种不饱和脂肪酸化合物。具体方案采用碱溶液对样品进行水解，酸化后直接通过萃取获得金松酸等脂肪酸的产物，通过一次逆流色谱分离便实现了香榧籽油中多种不饱和脂肪酸成分的分离纯化，有效地缩短了分离制备的时间；并且该方法不仅成本低，而且操作简单可靠，为香榧籽油中不饱和脂肪酸的开发利用提供了技术支撑，具有较好的应用价值。
附图说明
图1为实施例1分离的逆流色谱分离谱图；
图2为实施例1所得分离物的HPLC分析图；
图3为实施例2的逆流色谱分离图；
图4为实施例2分段产物的HPLC分析图。
具体实施方式
除非有特殊说明，本文中的术语或方法根据相关领域普通技术人员的认识理解或采用已有相关方法实现。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本公开而不用于限制本公开的范围。
下述实施所用的逆流色谱仪为江阴逆流科技有限公司生产的OptiChrome-300PLUS型号高速逆流色谱仪。所用香榧籽油、试剂均为市售产品。
实施例1：
(1)将10mL 0.5M KOH水溶液和1g香榧籽油(该香榧籽油中二十碳二烯酸、油酸、金松酸、亚油酸的含量分别约为7.3％、12.9％、17.5％、38.4％)加入反应容器中，70℃水浴回流反应2h后得反应物；向反应物中加适量盐酸调节反应物pH至3左右后，加入正庚烷萃取三次(正庚烷共50mL)，合并萃取液，用旋转蒸发仪浓缩得浓缩物；
(2)将体积比为10:9:1的庚烷、无水乙醇和水混匀后静置分层并分离出上层液体和下层液体，分别为上相和下相；在500mg的浓缩物中加入上相和下相溶液各5mL，将样品充分溶解得样品溶液；
(3)以15mL/min速度泵入上相溶液作为固定相，待固定相充满逆流色谱后，开启速度控制器，转速达到1000rpm以后以4mL/min的速度泵入下相，将样品溶液通过进样系统注入逆流色谱，开启紫外检测器，设定检测波长210nm，温度为30℃。逆流色谱分离谱图如图1所示。
收集洗脱时间为115-220分钟的分离物，并对分离物进行HPLC检测和核磁检测。
HPLC分析的条件为：色谱柱为YMC-C18色谱柱(id 4.6×150mm，5μm)；以甲醇(A)和三氟乙酸(0.03％，v/v)溶液(B)为流动相进行梯度洗脱； 所述梯度洗脱条件为0～15min，80％-100％A，15～25min，100％A；柱温为30℃；流速为0.8mL/min；进样体积为5μL；检测波长为210nm。图2为实施例1分离的游离脂肪酸的HPLC分析谱图。
进一步对各分离物进行核磁共振检测分析，鉴定结果如下：
115-158min时间段分离物：ESI-MS,m/z 599.35[2M+K] +。 1H-NMR(400MHz,MeOD)δ:5.37(m,4H),2.78(t,2H),2.30(t,2H),2.06(m,4H),1.64(m,2H),1.37(m,14H),0.90(m,3H)。 13C-NMR(100MHz,MeOD)δ：178.20,131.50，131,04，129.25，129.20，35.33，32.82,30.86,30.62，30.47，30.40，30.40，28.35，28.35，26.75，26.29，23.79，14.71。鉴定化合物为亚油酸。
165-179min时间段分离物：ESI-MS,m/z 651.31[2M+K] +。 1H-NMR(400MHz,MeOD)δ:5.34(m,6H),2.78(t,2H),2.30(t,2H),2.06(m,8H),1.64(m,2H),1.37(m,2H),1.31(m,8H),0.90(m,3H) 13C-NMR(100MHz,MeOD)δ：177.77,131.97,131.15,130.99,130.02,129.38,129.24,35.22,34.56,33.27,30.82,30.82,30.62,30.46,28.24,27.76,26.73,26.3,23.84,14.63。鉴定化合物为金松酸。
185-195min时间段分离物：ESI-MS,m/z 603.42[2M+K] +。 1H-NMR(400MHz,MeOD)δ:5.34(m,2H),2.30(t,2H),2.06(m,4H),1.64(m,2H),1.37(m,2H),1.31(m,18H),0.90(m,3H)。 13C-NMR(100MHz,MeOD)δ：177.58,130.80，130.70，34.89，33.00，30.78，30.73，30.42，30.39，30.28，30.25，30.17，30.13，28.06，28.05，26.03，23.67，14.39。鉴定化合物为油酸。
205-220min时间段分离物：ESI-MS,m/z 655.31[2M+K] +。 1H-NMR(400MHz,MeOD)δ:5.34(m,4H),2.78(t,2H),2.30(t,2H),2.06(m,6H),1.64(m,2H),1.31(m,16H),0.90(m,3H) 13C-NMR(100MHz,MeOD)δ：177.63，131.57，130.68，130.49，128.84，34.85，32.84，32.46，30.62，30.53，30.12,30.05,29.97，27.92，27.80，27.35，26.32，25.93，23.41，14.21。鉴定化合物为二十碳二烯酸。
实施例1的的分离结果如图1所示，在4h内完成分离，经HPLC分析后合并，得到高纯度的亚油酸、金松酸、油酸、二十碳二烯酸产物，分离得到的亚油酸、金松酸、油酸、二十碳二烯酸质量分别为：39mg、18mg、13mg、7mg；并采用面积归一化的方法计算样品纯度，分离得到的亚油酸、金松酸、油酸、二十碳二烯酸的纯度分别为：98％、96％、94％、96％。
实施例2：
该实施例与实施例1不同的是，用体积比为5:5:9:1正庚烷、乙酸乙酯、甲醇和水溶剂体系替换庚烷、无水乙醇和水溶剂体系；收集70-80min以及85-95min段分离产物。
图3为实施例2逆流色谱分离图；图4为实施例2逆流分段产物HPLC分析图，从图中可以看出4种脂肪酸没有得到有效的分离，分离产物为几种脂肪酸的混合物。

Claims (6)

  1. 一种香榧籽油中多种高纯度不饱和脂肪酸的分离方法，其特征在于，所述方法包括以下步骤：
    (1)将香榧籽油与碱性水溶液在40-80℃条件下回流反应，反应完成后用1M盐酸或冰乙酸调整反应物pH值为3-5后，用有机溶剂萃取pH值为3-5的所述反应物，所得萃取液经浓缩得浓缩物；所述碱性水溶液为KOH水溶液或NaOH水溶液；
    (2)所述浓缩物用上相和下相溶解后得样品溶液；庚烷、无水乙醇和水混合后静置分离上层液体和下层液体，其中上层液体为所述上相、下层液体为所述下相；
    (3)所述样品溶液用逆流色谱分离得多种不饱和脂肪酸，逆流色谱分离的固定相为所述上相，流动相为所述下相；流动相的泵送速度为1-10mL/min，检测波长为190-230nm，柱温为20-40℃；逆流色谱仪器转速为500-1500rpm。
  2. 如权利要求1所述的香榧籽油中多种高纯度不饱和脂肪酸的分离方法，其特征在于，流动相的所述泵送速度为4mL/min，所述检测波长为210nm，所述柱温为30℃，所述仪器转速为1000rpm，收集洗脱时间为115min-220min时间段的分离物得到多种不饱和脂肪酸。
  3. 如权利要求2所述的香榧籽油中多种高纯度不饱和脂肪酸的分离方法，其特征在于，分别收集洗脱时间为115-158min、165-179min、185-195min、205-220min时间段的分离物得到亚油酸、金松酸、油酸和二十碳二烯酸。
  4. 如权利要求1所述的香榧籽油中多种高纯度不饱和脂肪酸的分离方法，其特征在于，在步骤(1)中，所述有机溶剂选用庚烷、戊烷、己烷、石油醚或者乙酸乙酯。
  5. 如权利要求1所述的香榧籽油中多种高纯度不饱和脂肪酸的分离方法，其特征在于，在步骤(2)中，所述庚烷、无水乙醇和水按体积比10:9:1混合。
  6. 如权利要求1或权利要求5所述的香榧籽油中多种高纯度不饱和脂肪酸的分离方法，其特征在于，在步骤(2)中，所述庚烷、无水乙醇和水混合液替换为庚烷、甲醇和水的混合液，己烷、甲醇和水混合液或者己烷、无水乙醇和水混合液。
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