Preparative Isolation of High-Purity n-3 Docosapentaenoic Acid via Iterative Isocratic Flash Chromatography with Solvent Recycling

Background: n-3 Docosapentaenoic acid (DPA; 22:5 n-3) is increasingly viewed as a distinct long-chain omega-3 fatty acid with biological activities that are not fully captured by eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). However, progress remains limited by restricted access to high-purity DPA: most commercial sources contain DPA as a minor component, and published isolation strategies often yield only enriched mixtures or require multi-step workflows that are difficult to scale in standard laboratories. Objectives: We aimed to establish a robust, laboratory-accessible purification workflow to obtain DPA ethyl ester at high purity while preserving oxidative quality. Methods: Candidate lipid sources were screened to select an optimal DPA-containing feedstock. Oils were stabilized with antioxidants and pre-fractionated by cold crystallization (−20 °C) to reduce saturated lipids and oxidation by-products. Preparative separation used a stacked C18 flash system (15 μm + 45 μm in series) operated isocratically (methanol/water 92:8, v/v) at 120 mL/min. Fractions were analyzed by GC and iteratively reinjected to progressively enrich the DPA window. Solvent was recovered by distillation and reused. Results: Omegavie® 4020EE (5.4% n-3 DPA) was identified as the best starting material. Pretreatment eliminated detectable TBARS-derived malondialdehyde. The isocratic purification-loop strategy produced tens of grams of DPA ethyl ester at >98% purity (GC–FID) defined as n-3 DPA area% of total identified fatty acid methyl esters by GC–FID, with per-cycle DPA recovery of 91–95%, overall recovery of 76% from the starting DPA content, and >90% solvent recycling. The workflow is scalable at the gram-to-tens-of-grams level for research laboratories, although solvent burden and column maintenance remain practical constraints for larger-scale implementation. Identity and purity were confirmed by GC–MS and ^1H NMR, and oxidation indices remained low (peroxide value < 0.2 meq/kg; p-anisidine < 3). Conclusions: This scalable, solvent-conscious protocol enables reliable access to high-purity DPA and should be adaptable to other low-abundance polyunsaturated fatty acids.