Preparative and scaled-up separation of high-purity α-linolenic acid from perilla seed oil by conventional and pH-zone refining counter current chromatography.

α-Linolenic acid is an essential omega-3 fatty acid needed for human health. However, the isolation of high-purity α-linolenic acid from plant resources is challenging. The preparative separation methods of α-linolenic acid by both conventional and pH-zone refining counter current chromatography were firstly established in this work. The successful separation of α-linolenic acid by conventional counter current chromatography was achieved by the optimized solvent system n-heptane/methanol/ water/acetic acid (10:9:1:0.04, v/v), producing 466 mg of 98.98% α-linolenic acid from 900 mg free fatty acid sample prepared from perilla seed oil with linoleic acid and oleic acid as by-products. The scaled-up separation in 45× is efficient without loss of resolution and extension of separation time. The separation of α-linolenic acid by pH-zone refining counter current chromatography was also satisfactory by the solvent system n-hexane/methanol/water (10:5:5, v/v) and the optimized concentration of trifluoroacetic acid 30 mM and NH4 OH 10 mM. The separation can be scaled up in 180× producing 9676.7 mg of 92.79% α-linolenic acid from 18 000 mg free fatty acid sample. pH-zone refining counter current chromatography exhibits a great advantage over conventional counter current chromatography with 20× sample loading capacity on the same column.