Preparation Of High Purity EPA-EE And DHA-EE Using Supercritical CO₂

EPA and DHA have different effect on human health,such as anti-cancer and prevention of cardiovascular disease,and positive effects in the development of infant retina and brain.However,fish oil contains other components such as metal ion and fish oil oxide,which are harmful to human body.What's more,EPA and DHA play different role in treatment of disease,and the interaction is detrimental.So,it is of great importance to enrich EPA and DHA content to a desired level.High content EPA and DHA products have high market value.However,traditional enrichment methods,such as urea complexation and AgNO₃-complexation,cannot enrich EPA and DHA to more than 70%.Modern methods to prepare high purity EPA and DHA,such as high performance liquid chromatography,cannot increase content and output at the same time,and also the use of organic solvent can easily debase the quality of products.Supercritical CO₂ is widely used in extraction and enrichment of natural product for its high solubility and efficiency.In this paper,improved methods of supercritical CO₂ extraction were applied to enrich EPA-EE and DHA-EE in fish oil ethyl esters.The influence of process parameters were studied,and a new method to enrich EPA-EE and DHA-EE using supercritical CO₂ fractionation combined with column chromatography was developed,which greatly improved the content and output at the same time.A supercritical CO₂ fractionation strategy combined with stepwise increasing pressure was presented to concentrate DHA-EE in fish oil ethyl esters mixtures from 60%to 89.16%,and the influence of process parameters,such as temperature gradient and pressure,were studied,respectively.DHA-EE content increased with increasing temperature gradient due to increasing solubility difference.Moreover,DHA-EE could be concentrated by removing light and heavy part and keeping middle fractions through stepwise increasing pressure.A continuous countercurrent extraction strategy using supercritical CO₂ fractionation was presented to improve output,in which DHA-EE in fish oil ethyl esters mixtures was enriched from 60%to 77.31%,and the influence of process parameters,such as temperature gradient and pressure,were studied,respectively.DHA-EE could be concentrated under lower pressure and higher temperature gradient via multiple countercurrent extractions.What's more,the pilot scale test showed good stability.To further improve column efficiency,a supercritical CO₂fractionation apparatus combined with column chromatography was presented,using different kinds of raw materials.The effects of CO₂ mass flow rate,size and type of packing material,temperature gradient,pressure,column height and loaded amount were investigated,respectively.CO₂mass flow rate had little influence on DHA-EE content,for the mass transfer was mainly controlled by internal diffusion.Nevertheless,excessively fast CO₂ mass flow rate was harmful to the enrichment,due to short residence time between fluid and packing material.The combination between components with large number of double bond and packing material became stronger when the adsorptivity of packing material was high,while small particle size of packing material was not suitable for the enrichment.DHA-EE content increased with increasing temperature gradient,and was higher under specific pressure.EPA-EE and DHA-EE content increased with increasing column height,due to increasing mass transfer between components and packing materials,as well as supercritical CO₂.Also EPA-EE and DHA-EE content decreased with higher loaded amount,which was due to the backmixing of components.The highest content of DHA-EE in samples obtained was 92.02%in DHA-EE sample,while the highest content of EPA-EE and DHA-EE in samples obtained was 85.34%in EPA-EE sample.The newly developed supercritical CO₂ fractionation method,in which no pretreatment of raw materials as well as organic solvents were used,not only greatly improved the content and output,but also simplified the operation procedure and kept the quality of nature products,which is a feasible method for EPA-EE and DHA-EE enrichment.