| ?-linolenic acid(ALA),an essential omega-3 fatty acid needed for human health.It provides cardiovascular protection,anticancer,neuroprotection,anti-osteoporosis,anti-inflammatory and anti-oxidant effects.Perilla seed oil contains a large amount of unsaturated fatty acids,of which ALA content might up to 60%.Although the traditional separation and purification methods can obtain high purity fatty acids,certain defects ouccrs when those methods applied to ?-linolenic acid purification,such as incomplete separation,low yield,high loss,and low purity.Countercurrent chromatography has been used in purification applications widely,since its flexible and fast operation,high efficiency,large preparation amount and low cost.Compared with conventional countercurrent chromatography,pH-zone-refining countercurrent chromatography is more suitable for separation of acidic or alkaline substances.In this paper,?-linolenic acid separation from perilla seed oil by both conventional countercurrent chromatography and pH-zone-refining countercurrent chromatography were established,which provided new methods and technical supports for efficient separation and preparation of ?-linolenic acid from perilla oil.The main research contents include:1.Establishment of analytical method for ?-linolenic acidFirstly,HPLC-DAD and GC-MS were used to analyze and detect ?-linolenic acid and other fatty acids in perilla seed oil.Without pretreatment,HPLC-DAD directly detected fatty acids for good reproducibility,short analysis time,fast and easy operation.GC-MS provided good chromatographic peak resolution,high peak capacity and high detection sensitivity for unsaturated fatty acids.However,it was a time-consuming analysis,and methyl esterification for fatty acid is required.HPLC-DAD was used to determine the K value of alternative solvent systems for conventional countercurrent chromatography and pH-zone-refining countercurrent chromatography.It was also used for the detection of separated fractions,determination of fractions and elution curves drawing.GC-MS was used to qualitatively and quantitatively analyze components of perilla seed oil sample,as well as purity of the final product ?-linolenic acid.2.Separation of ?-linolenic acid by conventional CCCBy selection of different proportions of n-heptane : methanol : water solvent system with adding appropriate amount of acetic acid,the optimal solvent system was finally determined to be n-heptane : methanol : water : acetic acid = 10 : 9 : 1 : 0.04(v/v).The separation and scale-up experiments of conventional countercurrent chromatography were carried out using the optimal solvent system.The volume ratio of the column was taken as the scale-up ratio,therefore,the injection volume and flow phase elution flow rate were multiplied by this ratio.The analytical conventional countercurrent chromatography was used to purify 20 mg perilla seed oil and obtained 9.8 mg of ?-linolenic acid with purity of 98.43 %.The semi-preparative conventional countercurrent chromatography was used to purify 120 mg perilla seed oil and obtained 60.5 mg of ?-linolenic acid with purity of 98.32 %.The preparative conventional countercurrent chromatography was used to purify 900 mg perilla seed oil and obtained 466.4 mg of ?-linolenic acid with purity of 98.98 %.During the separation and scale up process,baseline separation was achieved between ?-linolenic acid,linoleic acid and oleic acid constantly.Without prolonging the separation time,the sample loading was scaled up 45 times totally.3.Separation of ?-linolenic acid by pH-zone-refining CCCFirst,keeping concentration of retainer acid and eluter base(CTFA : CNH3.H2O = 30 mM : 20 mM),different proportions of n-hexane : methanol : water solvent system were selected.Finally,n-hexane : methanol : water = 10 : 5 : 5(v/v)was selected as the basic solvent system.The concentration and proportion of retainer acid and eluter base were optimized on the basis of the selected solvent system.Final optimized solvent system for ?-linolenic acid separation by pH-zone-refining countercurrent chromatography was n-hexane : methanol : water = 10: 5: 5(v/v),and the concentration of retainer acid and eluter base was CTFA : CNH3.H2 O = 30 mM : 10 mM.The ?-linolenic acid was separated and scaled-up under the optimal solvent system.9000 mg perilla seed oil were purified by preparative pH-zone-refining countercurrent chromatography and produced 4780.2 mg ?-linolenic acid with purity of 93.09 %.After a further optimization of the concentration of retainer acid and eluter base,9676.7 mg ?-linolenic acid with purity of 92.79 % was obtained by one-time separation from 18000 mg sample injection.Compared to preparative conventional countercurrent chromatography,the separation capacity by pH-zone-refining CCC was further improved by 20 times.This successful scale up fully reflected the unique advantages of pH-zone-refining countercurrent chromatography in acidic compounds separation. |
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