AU2008283113A1 - Use of ionic liquids for extraction or fractionation of lipids - Google Patents

Description

WO 2009/017425 PCT/NZ2008/000183 1 USE OF IONIC LIQUIDS FOR EXTRACTION OR FRACTIONATION OF LIPIDS TECHNICAL FIELD 5 This invention relates to separation technology. In particular, the invention relates to a process which employs an ionic liquid for extracting unsaturated lipophilic compounds from lipid containing feed materials. The process is particularly useful for extracting polyunsaturated fatty acids, or their derivatives, from feed materials containing a mixture of fatty acids or their derivatives. 10 BACKGROUND There is a substantial body of research which demonstrates the beneficial effects of polyunsaturated fatty acid (PUFA) consumption in the prevention and/or treatment of a 15 variety of diseases including cardiovascular conditions, inflammatory diseases and some tumours [1,2]. Therefore there is a demand for PUFA and their derivatives for use as, or in, dietary supplements and pharmaceuticals. Many methods for the separation of unsaturated fats and fat derivatives from saturated 20 fats and fat derivatives have been reported, but only a few are industrially useful for large-scale production. Technologies that have been employed industrially include adsorption chromatography, fractional or molecular distillation, enzymatic splitting, low temperature crystallization, urea complexation and supercritical fluid extraction/fractionation. Separation of unsaturated fats or fat derivatives from saturated 25 fats and fat derivatives using these methods is complicated by several factors. Firstly, methods relying on differences in physical properties, such as molecular weight, are hindered because the physical properties of the unsaturated and saturated fats are very similar [3], especially when attempting to separate saturated and unsaturated fatty acids of the same chain length. Secondly, the unsaturated fatty acids are readily susceptible 30 to oxidation, degradation, polymerization and stereomutation, even at moderately elevated temperatures. Oxidative deterioration in particular is problematic since the primary oxidation products (lipid hydroperoxides) are unstable and can further degrade to yield volatile secondary oxidation products. These can impart unpleasant fishy odours and flavours to end products, which is particularly undesirable if the products are 35 for human consumption.

WO 2009/017425 PCT/NZ2008/000183 2 The concentration of the unsaturated lipids in the form of triglycerides (TAG) is more difficult because the fatty acids are randomly arranged on the glycerol backbone of the TAG [3]. Therefore, the fat or oil is usually converted into free fatty acids (FFA) or fatty acid ethyl esters (FAEE) before separation into polyunsaturated and saturated fractions 5 is carried out. Most separation methods, when used alone, can only separate fatty acids into group fractions. Therefore, two or more processes are required to produce concentrated polyunsaturates. The production of individual polyunsaturated components in high purity requires further processing steps. .10 One of the most effective methods for separating saturated and unsaturated long-chain molecules is urea fractionation. The use of urea complexes to separate saturated and monounsaturated fatty acids from PUFA has been known since the 1950s [4]. The stability of the complexes formed between urea and FFA is highly dependent on the degree of unsaturation in the fatty acids, with saturated fatty acids forming the most 15 stable complexes and PUFA forming the least stable. The solid urea complexes are removed from the PUFA-containing solution by filtration. The PUFA are recovered from the filtrate by solvent extraction with a non-polar organic solvent, such as hexane or isooctane. Large quantities of urea and large volumes of organic solvent are often required for this process, which is undesirable given the health and safety and 20 environmental considerations governing today's chemical and manufacturing industries. Organic solvent solutions containing silver ions, have also been used in extraction [5] and chromatographic [6, 7] processes to separate saturated and monounsaturated fatty acids or esters from PUFA. Here, the silver ion forms weak n-bonds with sites of unsaturation in lipids. The strength of the bonds increases with the number of sites of 25 unsaturation, and hence PUFA are preferentially extracted from a mixture. However, silver ions (from dissolution of AgNO 3 ) are highly toxic and unsuitable for use when the final end product is for human consumption; the silver ions are very expensive, the silver ions are gradually leached from solution or the chromatographic packing, and the silver ions are easily decomposed by light to an insoluble salt that has no activity, rendering 30 the processes inoperable. Supercritical fluids are selective solvents that have found application in various extraction processes, including the isolation of PUFA and other multiply unsaturated lipids from lipid-containing feed materials [8-10]. Supercritical fluid technologies can 35 overcome some of the problems associated with the traditional processing methods (use of lower temperatures, reduced quantities of organic solvent, and extraction under an WO 2009/017425 PCT/NZ2008/000183 3 inert atmosphere; often carbon dioxide), but specialized equipment is required and low throughput (often due to solubility limitations) can greatly increase processing costs. In addition, supercritical fluids show little to no selectivity between fatty acids or fatty acid derivatives of similar chain length but different degrees of unsaturation, and so must be 5 used along with another process to concentrate polyunsaturates. Ionic liquids have received a great deal of attention as reaction and extraction media because their novel solvent properties offer several advantages over conventional organic solvents. They are solvents for a wide range of inorganic and organic materials 10 and their solvent power can be tuned by careful selection of the constituent cations and anions. Ionic liquids have no vapour pressure thus eliminating the emission of volatile organics into the atmosphere. Moreover, most are thermally stable at > 450 K, and they have a large liquid range enabling tremendous kinetic control over chemical processes. Ionic liquids are miscible with substances having a very wide range of polarities and they 15 can simultaneously dissolve organic and inorganic materials. These properties offer numerous opportunities for the modification of existing and/or for the development of new extraction processes. Several researchers have reported using ionic liquids for the separation of gasses, 20 saturated and unsaturated volatile hydrocarbons and aromatic ring compounds. There are no reports of their use in conjunction with near-critical fluids for the production of polyunsaturated lipid-rich or pure polyunsaturated lipid fractions. GB 2,371,805 describes the separation of volatile olefins from non-olefins using ionic 25 liquids and ionic liquid solutions. The processes described use transition metal salts, such as silver salts, dissolved or suspended in ionic liquids to preferably separate mono olefins from non-olefins including paraffins, oxygenates and/or aromatic compounds. The olefins are preferentially extracted into the ionic liquid/metal salt suspension/solution. However, the use of metal salts, e.g. silver salts, greatly increases 30 the processing cost and is undesirable for the production of end products for human consumption, e.g. human dietary supplements. WO 03/059483 describes the use of ionic liquids for separating volatile di-olefins from mono-olefins. Again, ionic liquid/metal salt media are used to facilitate the separation. 35 US 2005/0150383 and US 2005/0154247 describe using ionic liquids and metal salts supported on porous transport membranes to facilitate the separation of volatile olefins WO 2009/017425 PCT/NZ2008/000183 4 from non-olefins. Again, the use of metal salts is undesirable for the production of products for human consumption and the use of membranes increases the complexity and cost of the process. 5 EP 1476408 describes the use of ionic liquids for the separation of water-soluble organic compounds. The extracting media can comprise solely of an ionic liquid or an ionic liquid with additional components selected from hydrocarbons, alcohols, carboxylic acids, carboxylic esters, lactones, lactams, amides, nitriles, carbonates, amines and water. The separation processes described are limited to water-soluble species, and 10 thus are not suitable for the extraction of polyunsaturated lipids, which are not water soluble. The separation of polyunsaturated lipid components is not described. WO 2006/012513 and a related journal article [11] describe di-ionic liquids. The di-ionic liquids consist of two singly-charged ionic liquids linked by a bridging group, along with 15 either two singly charged counter ions or one doubly charged counter ion. The bridging group also contains functional groups that enable the di-ionic liquid to be immobilised on solid materials such as gas chromatographic stationary phases, and may also allow cross linking by polymerisation if the bridging group has sites of unsaturation. An example of the use of these di-ionic liquids immobilised on a gas chromatography 20 column is given in which C 6

-C

24 straight-chain fully saturated fatty acid methyl esters are separated by gas chromatography at temperatures of 373 to 533 K. However, the process described is clearly not applicable to the separation of polyunsaturated fatty acids because the high temperatures employed would prevent the formation of weak bonds between the cation and sites of unsaturation that enable polyunsaturates to be 25 extracted or separated. Li and Li [12] describe the enrichment of omega-3 PUFA fatty acid methyl esters from cod liver oil using 1-hexyl-3-methyl imidazolium hexafluorophosphate containing silver salts. The silver ions are described as being important for separating the 30 polyunsaturated methyl esters, and the ionic liquid simply provides a solution in which the silver ion is not readily leached or decomposed. The use of supercritical fluids to recover the PUFA after fractionation is not described. The authors comment that the process must be performed in the absence of light, and show that AgBF 4 is preferred over AgNO 3 as the source of silver ions. The use of silver ions is less desirable for the 35 production of an end-product for human consumption, as well as from a cost perspective.

WO 2009/017425 PCT/NZ2008/000183 5 The present invention concerns the use of phases containing ionic liquids for the extraction of unsaturated lipids from lipid-containing feed materials at temperatures less than 373 K. Ionic liquid extraction can be used as an alternative low-temperature 5 method for unsaturated lipid (particularly PUFA) extraction before subsequent recovery of the unsaturated lipid by solvent or supercritical fluid extraction. The present invention also concerns the extraction of unsaturated lipids from a supercritical fluid phase by an ionic liquid. Combining ionic liquids with supercritical fluid extraction eliminates the need for organic solvents and provides solvent residue-free extracts with wide consumer 10 appeal. It is therefore an object of the present invention to provide a process for extracting unsaturated lipids from feed materials containing lipids using ionic liquids, which goes some way towards overcoming the above disadvantages, or at least provides a useful 15 choice. STATEMENTS OF INVENTION In a first aspect the invention provides a process for separating one or more unsaturated 20 lipids from a feed material, comprising at least the steps of: (i) contacting the feed material with an ionic liquid phase to give: a. an ionic liquid phase containing one or more. unsaturated lipids; and b. a raffinate; 25 (ii) separating the ionic liquid phase containing one or more unsaturated lipids from the raffinate; and (iii) recovering one or more unsaturated lipids from the ionic liquid phase containing one or more unsaturated lipids. 30 In a second aspect the invention provides a process for separating one or more unsaturated lipids from a feed material, comprising at least the steps of: (i) contacting the feed material with a near-critical fluid to give: a. a near-critical solution containing one or more unsaturated lipids; and 35 b. a raffinate; (ii) separating the near-critical solution from the raffinate; WO 2009/017425 PCT/NZ2008/000183 6 (iii) contacting the near-critical solution with an ionic liquid phase to give: c. an ionic liquid phase containing one or more unsaturated lipids; and d. a lipid-depleted near-critical fluid phase; 5 (iv) separating the ionic liquid phase containing one or more unsaturated lipids from the lipid-depleted near-critical fluid phase; and (v) recovering the one or more unsaturated lipids from the ionic liquid phase containing one or more unsaturated lipids. 10 Preferably the raffinate is liquid. Preferably one or more of the unsaturated lipids are polyunsaturated fatty acids (PUFA) or fatty acid derivatives. The fatty acid derivatives are not limited to any particular fatty acid derivatives, but preferred fatty acid derivatives may be selected from the group 15 comprising fatty acid C 1

-C

4 alcohol esters, fatty acid amines, fatty acid ethanolamines, fatty acid amides, and fatty alcohols. The PUFA are preferably selected from cis-5,8,11,14,17-eicosapentaenoic acid, cis 4,7,10,13,16,19-docosahexaenoic acid, 6,9,12-octadecatrienoic acid (y-linolenic acid), 20 9,12,15-octadecatrienoic acid (a-linolenic acid), 9,11-octadecadienoic acid (conjugated linoleic acid), and esters thereof. The one or more unsaturated lipids are preferably selected from the group comprising carotenoids, partial glycerides, triglycerides, hydrocarbons, alkyldiacyl glycerides, fat 25 soluble vitamins, tocotrienols, phospholipids, glycolipids, and sphingolipids. It is preferred that the ionic liquid used in the process of the invention comprises a cation selected from the group comprising substituted-quinolinium [R 1

.

10 Chin]*, substituted imidazolium [R- 5 1M]*, substituted-pyridinium [R 1 ePy]*, substituted-ammonium [Rl4N]*, 30 substituted-phosphonium [R 14 P]*, substituted-pyrrolidinium [R 1

-

1 0 Pyrr]*, where each R is hydrogen, alkyl, substituted-alkyl, alkoxy, substituted-alkoxy, aryl or substituted-aryl, or combinations thereof. Preferably the cation is a substituted-pyrrolidinium, substituted imidazolium, substituted pyridinium, or substituted quinolinium. 35 In preferred embodiments of the invention ionic liquid comprises an anion selected from the group comprising acetate [OAc]-, bis[1,2-benzenediolato(2-)-O,O'1-borate [BBB]-, WO 2009/017425 PCT/NZ2008/000183 7 tetracyanoborate [B(CN) 4 1-, tetrafluoroborate [BF4]-, bis(methylsulfonyl)imide [BMA]-, bis(malonato(2-))borate [BMB]-, bis(oxalate(2-))borate [BOB]-, bis(salicylato(2-))borate [BSB]-, bis(trifluoromethylsulfonyl)imide [BTA]~, trifluoromethylsulfonate [CF 3

SO

3 ], methylsulfonate [CH 3

SO

3 ]~, methylsulfate [CH 3

SO

4 1-, ethylsulfate [C 2

H

5 SO4]-, 5 propylsulfate [C 3

H

7 SO4]-, butylsulfate [C 4

H

9 SO4], pentylsulfate [C 5

H

1 S0 4 1-, hexylsulfate

[C

6

H

1 3

SO

4 ]-, heptylsulfate [C 7

H

15

SO

4 ], octylsulfate [C 8

H

1 7 SO41, chloride [Cl]-, dimethylphosphate [DMPO 4 ]-, hydrogensulfate [HSO 4 ]-, N-methylsulfonylacetamide [MAcA]-, 2-(2-methoxyethoxy)ethylsulfate [MDEGSO4]-, dicyanamide [N(CN) 2 ] , hexafluorophosphate [PF6]-, thiocyanate [SCN]-, p-toluenesulfonate [TOS]-, and 10 salicylate [Sal]-. Examples of the ionic liquid used in the process include, but are not limited to, 1-ethyl-3 methylimidazolium dicyanamide, 1-butyl-3-methylimidazolium dicyanamide, 1-butyl-3 methylimidazoilium trifluoromethanesulfonate, 1-butyl-3-methylimidazoilium 15 hexafluorophosphate, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, 1 hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, 1-ethyl-3-methylpyridinium bis(trifluoromethylsulfonyl)imide, and trioctylmethylammonium bis(trifluoromethylsulfonyl)imide. 20 Preferably the feed material is a lipid containing material of, or derived from, marine, plant or animal origin. Preferably the ionic liquid phase consists of a single ionic liquid or a mixture of ionic liquids. 25 In certain embodiments of the invention the ionic liquid phase in step (i) of the first aspect of the invention may be a mixture of an ionic liquid and a near-critical fluid. The pressure of the near-critical fluid may be increased to recover the unsaturated lipids from the ionic liquid phase in step (iii) of the first aspect of the invention. The pressure of 30 the near-critical fluid may be increased in incremental steps to give one or more lipid containing fractions, where when more than one fraction is obtained, the fractions do not have the same lipid composition. For example, the different fractions may contain lipids which vary in the degree of unsaturation of the lipids and/or composition in terms of lipid types. 35 WO 2009/017425 PCT/NZ2008/000183 8 In other embodiments the ionic liquid phase may be a mixture of an ionic liquid and an organic solvent or a mixture of organic solvents. In some preferred embodiments of the invention, contacting the feed material with an 5 ionic liquid phase according to step (i) of the first aspect of the invention comprises: (a) contacting the ionic liquid with a near-critical fluid to give a near-critical fluid-containing ionic liquid phase; and (b) contacting the feed material with the near-critical fluid-containing ionic liquid phase to give: 10 i. a near-critical fluid-containing ionic liquid solution phase containing one or more unsaturated lipids; and ii. a raffinate. In other preferred embodiments of the invention, contacting the feed material with an 15 ionic liquid phase according to step (i) of the first aspect of the invention comprises: (a) contacting the ionic liquid with the feed material to give: i. an ionic liquid phase containing one or more unsaturated lipids; and ii. a raffinate; and (b) contacting the ionic liquid phase and the raffinate with a near-critical 20 fluid to give: iii. a near-critical fluid-containing ionic liquid solution phase containing one or more unsaturated lipids; and iv. a raffinate. 25 Preferably the near-critical fluid-containing ionic liquid solution phase is separated from the raffinate, and one or more unsaturated lipids are recovered from the near-critical fluid-containing ionic liquid solution phase. In some preferred embodiments of the invention recovering the one or more unsaturated 30 lipids according to step (iii) of the first aspect or step (v) of the second aspect of the invention comprises: (a) contacting the ionic liquid phase containing one or more unsaturated lipids with a near-critical fluid to extract the one or more unsaturated lipids from the ionic liquid phase to give a near-critical solution of one 35 or more unsaturated lipids; and WO 2009/017425 PCT/NZ2008/000183 9 (b) recovering the one or more unsaturated lipids from the near-critical solution by pressure reduction and/or temperature change to reduce the solvent power of the near-critical fluid. 5 It is preferred that the ionic liquid phase containing one or more unsaturated lipids is in the form of a solution. Alternatively, the ionic liquid may be a solid-supported ionic liquid and the ionic liquid phase containing one or more unsaturated lipids is in the form of one or more unsaturated lipids adsorbed to the solid-supported ionic liquid. 10 DETAILED DESCRIPTION Definitions The term "unsaturated lipid" as used herein refers to constituents of fats and oils that contain one or more sites of unsaturation in the molecule. These may occur in the fatty 15 acid portions of the molecule such as in triglycerides, phospholipids and glycolipids, or in alkyl chains in the molecule, such as in carotenoids, hydrocarbons and fat soluble vitamins. The term "fatty acid(s)" as used herein refers to long-chain carboxylic acids and their 20 derivatives. Examples of fatty acid derivatives include, but are not limited to, fatty acid

C-C

4 alcohol esters, fatty acid amines, fatty acid ethanolamines, monoglycerides, fatty acid amides and fatty alcohols. The carbon chain can be branched or unbranched and can be saturated or unsaturated. Preferably the carbon chain is unbranched and contains from 4 to 24 carbon atoms. More preferably the carbon chain contains from 12 25 to 24 carbon atoms. The term "PUFA" as used herein refers to a polyunsaturated fatty acid, meaning a fatty acid having multiple double bonds, typically 2 to 6 double bonds. 30 The term "SFA" as used herein refers to a saturated fatty acid, meaning a fatty acid containing no double or triple bonds. The term "MUFA" as used herein refers to a monounsaturated fatty acid, meaning a fatty acid having a single double bond. 35 WO 2009/017425 PCT/NZ2008/000183 10 The term "ionic liquid" as used herein refers to compounds which comprise at least one cation and at least one anion. The ionic liquid should be in the liquid state under the extraction conditions used. Preferably the ionic liquid is liquid at temperatures below 373 K at atmospheric pressure. More preferably the ionic liquid is liquid at, or close to, 5 room temperature at atmospheric pressure. A common term for these compounds is "room temperature ionic liquids". Ionic liquids are also commonly referred to as "molten salts". Ionic liquids can be bought commercially or can be synthesised by techniques well known to those skilled in the art of synthetic chemistry. 10 The term "contact" or "contacting" as used herein generally means admixing the feed materials containing the lipophilic compounds with the phase containing the ionic liquid, or admixing the feed materials containing the lipophilic compounds with the near-critical fluid, or admixing the phase containing the ionic liquid with the near-critical fluid in a suitable vessel using suitable apparatus as are well known in the art. When the phase 15 containing the ionic liquid is a liquid, suitable apparatus includes, but is not limited to, a stirred tank, a static mixer, a nozzle, a mixing valve, a packed column (packing can be structured or random), and concentric pipes. When the phase containing the ionic liquid is a solid, such as a solid-supported ionic liquid, suitable apparatus includes, but is not limited to, a packed column, a thin film column, a membrane contactor, and a stirred 20 tank. Preferably the ionic liquid phase, when liquid, and the lipophilic containing materials are contacted using a stirred tank or packed column apparatus. The term "solid-supported ionic liquids" as used herein refers to any ionic liquids supported on a suitable solid material. The ionic liquids may be absorbed, adsorbed, or 25 immobilised on the solid support material, which can include, but is not limited to, adsorbent particles, resins, chromatographic packings, membranes, and thin films. The term "immobilised" means ionically or covalently bound. The term "raffinate" as used herein refers to the remaining liquid or solid portion of any 30 liquid or solid feed material after one or more unsaturated lipids have been extracted from that feed. The term "microporous" as used herein refers to materials having pore widths less than or equal to 2 nanometres. 35 WO 2009/017425 PCT/NZ2008/000183 11 The term "mesoporous" as used herein refers to materials having pore widths in the range 2 to 50 nanometres. The term "macroporous" as used herein refers to materials having pore widths greater 5 than 50 nanometres. Ionic Liquids The properties of ionic liquids can be tailored according to their intended use. This can be achieved by varying the combination of the cation and anion of the ionic liquid. 10 Examples of this are given in [13-17]. Preferred ionic liquids are those which are air and water stable. The ionic liquids can be neutral, basic or acidic. Neutral ionic liquids should be used to prevent the possibility of the polyunsaturated lipids being isomerised. 15 The cations may be singly or multiply charged organic species. Examples of cations for preparing ionic liquids include, but are not limited to: [R 1

.

1 oChin]*, substituted quinolinium; [R 1 .slM]*, substituted-imidazolium; [R 1 .ePy]*, substituted-pyridinium; [R 1 4N]*, substituted-ammonium; [R 1 4P]*, substituted-phosphonium; [R 1

-

1 oPyrr]*, substituted 20 pyrrolidinium, where each R is hydrogen, alkyl, substituted-alkyl, alkoxy, substituted alkoxy, aryl or substituted-aryl, or combinations thereof. Preferred ionic liquids are singly charged and are those containing nitrogen-substituted cations. More preferably, the cation has a delocalised charge on a ring system. Single or multi-cyclic ring systems can be used. Preferably rings are 5 or 6-membered. Examples of preferred cation 25 cyclic systems include, but are not limited to, substituted-pyrrolidinium based cations, substituted imidazolium based cations, substituted pyridinium based cations, and substituted quinolinium based cations. R7 R6 R5

R

8 R9 + R N R 3 / I--, 30 Substituted-pyrrolidinium, where R 1

.R

1 O are each hydrogen, alkyl, substituted-alkyl, alkoxy, substituted-alkoxy, aryl or substituted-aryl, or combinations thereof.

WO 2009/017425 PCT/NZ2008/000183 12 R2

N-R

3 Substituted-imidazolium, where R 1

-R

5 are each hydrogen, alkyl, substituted-alkyl, 5 alkoxy, substituted-alkoxy, aryl or substituted-aryl, or combinations thereof. R4 R R3 NR1 R2 Substituted-pyridinium, where R 1

-R

6 are each hydrogen, alkyl, substituted-alkyl, alkoxy, substituted-alkoxy, aryl or substituted-aryl, or combinations thereof. 10 R, R 1 R10 R8 N R R7

R

3

R

5

R

6 R 4 Substituted-quinolinium, where R-R 1 0 are each hydrogen, alkyl, substituted-alkyl, alkoxy, substituted-alkoxy, aryl or substituted-aryl, or combinations thereof. 15 Example anions for preparing ionic liquids include but are not limited to: [OAc]~, acetate; [BBB]~, bis[1,2-benzenediolato(2-)-O,O']-borate; [B(CN)4]-, tetracyanoborate; [BF 4 ] , tetrafluoroborate; [BMA], bis(methylsulfonyl)imide; [BMB]-, bis(malonato(2-))borate; [BOB], bis(oxalate(2-))borate; [BSB]-, bis(salicylato(2-))borate; [BTA]~, 20 bis(trifluoromethylsulfonyl)imide; [CF 3

SO

3 ]~, trifluoromethylsulfonate; [CH 3

SO

3 ]-, methylsulfonate; [CH 3

SO

4 ], methylsulfate; [C 2

H

5

SO

4 ]~, ethylsulfate; [C 3

H

7 SO4]~, propylsulfate; [C 4

H

9

SO

4 ], butylsulfate; [C 5

H

11

SO

4 ], pentylsulfate; [C 6

H

1 3SO4]-, WO 2009/017425 PCT/NZ2008/000183 13 hexylsulfate; [C 7 H1 5

SO

4 ], heptylsulfate; [C 8

H

17 SO41, octylsulfate; [Cl]-, chloride; [DMP04], dimethylphosphate; [HSO4]-, hydrogensulfate; [MAcA]~, N methylsulfonylacetamide; [MDEGSO 4 ], 2-(2-methoxyethoxy)ethylsulfate; [N(CN) 2 1, dicyanamide; [PF 6 ]~, hexafluorophosphate; [SCN]-, thiocyanate; [TOS]-, p 5 toluenesulfonate; [Sal]-, salicylate. Preferably, the anion should have some degree of sterical shielding around the charge centre. Examples of anions with sterical shielding include, but are not limited to, hexafluorophosphate, [PF 6 ]*; bis(oxalate(2-))borate, [f30B]-; tetracyanoborate, [B(CN)4]-; bis(trifluoromethylsulfonyl)imide, [BTA]-; trifluoromethylsulfonate, [CF 3

SO

3 ]-; and bis(malonato(2-))borate, [BMB]~. 10 Discussion of Invention In a first aspect the invention provides a process for separating one or more unsaturated lipids from a feed material, comprising at least the steps of: (i) contacting the feed material with an ionic liquid phase to give: 15 a. an ionic liquid phase containing one or more unsaturated lipids; and b. a raffinate; (ii) separating the ionic liquid phase containing one or more unsaturated lipids from the raffinate; and (iii) recovering one or more unsaturated lipids from the ionic -liquid phase 20 containing one or more unsaturated lipids. The feed material can be a liquid or solid mixture of lipophilic compounds and the raffinate can be liquid or solid. Preferably the feed material is a liquid mixture of lipophilic compounds. It is also preferred that the raffinate is liquid. More preferably the feed 25 material is a liquid mixture of fatty acids or their derivatives and the raffinate is liquid. Mixtures of fatty acids typically comprise fatty acids of varying chain length and varying degrees of unsaturation, and are routinely categorised as saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). 30 The lipophilic compounds of interest for the present invention are preferably fatty acids or their derivatives. More preferably, the lipophilic compounds of interest are PUFA such as all cis-5,8,11,14,17-eicosapentaenoic acid (20:5w3 or EPA), all cis 4,7,10,13,16,19-docosahexaenoic acid (22:6w3 or DHA), 6,9,12-octadecatrienoic acid (y-linolenic acid, or GLA), 9,12,15-octadecatrienoic acid (a-linolenic acid or ALA), 9,11 35 octadecadienoic acid (conjugated linoleic acid or CLA) and esters thereof.

WO 2009/017425 PCT/NZ2008/000183 14 In one embodiment of the invention, the ionic liquid phase consists only of the ionic liquid or a mixture of ionic liquids, i.e. is not used with a solvent such as water or ethanol, and the lipids are recovered from the ionic liquid using a near-critical fluid in which the ionic liquid is not soluble. Therefore, the ionic liquid phase can easily be 5 recycled. Recycling of solvents in other processes presents complex technical difficulties and significant additional cost. Most other processes use large volumes of organic solvents which must either be recycled or disposed of at great expense. The use of ionic liquids is also environmentally beneficial because they have negligible vapour pressure and therefore do not release volatile compounds into the atmosphere. 10 Further, the use of ionic liquids for separating unsaturated lipids can be a simple three step operation (contact the unsaturated lipid containing feed material with ionic liquid, separate the ionic liquid solution from the raffinate and recover the unsaturated lipid from the ionic liquid using a near-critical fluid) whereas conventional processes are often complex multi-step operations. 15 In some preferred embodiments, contacting the feed material with an ionic liquid phase according to step (i) of the first aspect of the invention comprises: (a) contacting an ionic liquid with a near-critical fluid to give a near-critical fluid containing ionic liquid phase; and 20 (b) contacting the feed material with the near-critical fluid-containing ionic liquid phase to give: i. a near-critical fluid-containing ionic liquid solution phase containing one or more unsaturated lipids; and ii. a raffinate. 25 Steps (ii) and (iii) of the first aspect then follow. In other preferred embodiments of the invention, contacting the feed material with an ionic liquid phase according to step (i) of the first aspect of the invention comprises: 30 (a) contacting the ionic liquid with the feed material to give: i. an ionic liquid phase containing one or more unsaturated lipids; and ii. a raffinate; and (b) contacting the ionic liquid phase and the raffinate with a near-critical fluid to give: 35 v. a near-critical fluid-containing ionic liquid solution phase containing one or more unsaturated lipids; and WO 2009/017425 PCT/NZ2008/000183 15 vi. a raffinate. Steps (ii) and (iii) of the first aspect then follow. 5 The recovery of unsaturated lipids from the ionic liquid to produce the final unsaturated lipid rich fractions may be performed by any suitable means. A preferred recovery method is the use of subcritical or supercritical fluids. Every substance has its own "critical point" at which the liquid and vapour state of the 10 substance becomes identical. The term "supercritical" as used herein refers to the pressure-temperature region above the critical point of a substance. The term "subcritical" as used herein refers to the pressure-temperature region equal to or above the vapour pressure of the substance, but below the critical temperature. The term "near-critical" encompasses both the sub and supercritical regions. 15 The near-critical fluid used in the present invention to recover unsaturated lipids from the ionic liquid can be any fluid suitable for the purpose. Example near-critical fluids useful for the invention include, but are not limited to, carbon dioxide, dimethyl ether, partially to fully fluorinated analogues. of dimethyl ether, ethane, ethylene, propane, 20 difluoromethane, trifluoromethane, 1,1,1-trifluoroethane and 1,1,1,2-tetrafluoroethane. The ionic liquid phase containing the unsaturated lipid is contacted with the appropriate near-critical fluid. The unsaturated lipid is extracted from the ionic liquid phase to produce an unsaturated lipid-containing near-critical solution, leaving the ionic liquid phase to be reused. The unsaturated lipid can be recovered from the near-critical fluid 25 by a simple pressure reduction and/or temperature change step. The choice of the near critical fluid will be dictated by the types of lipid to be extracted from the ionic liquid. Preferably the near-critical fluid is selected from carbon dioxide or dimethyl ether. More preferably the near-critical fluid used is carbon dioxide. Because the solvent properties of the near-critical fluid are highly dependent on temperature and pressure, it is possible 30 to use the near-critical fluid both as a solvent to extract PUFA from an ionic liquid phase at certain (high solvency) pressure and temperature conditions, and to use an ionic liquid to extract PUFA from a near-critical phase at certain (low solvency) pressure and temperature conditions. 35 A near-critical fluid may also be used as part of the ionic liquid phase during the contacting step with the feed material. The near-critical fluid can reduce the viscosity of WO 2009/017425 PCT/NZ2008/000183 16 the ionic liquid, which facilitates better mixing to aid the dissolution of the unsaturated lipids. Introducing a lipophilic near-critical fluid into the ionic liquid phase during the feed contacting step can also enhance the lipophilic nature of the ionic liquid phase, thus enhancing the solubility of the lipids in the ionic liquid phase over the solubility in the 5 ionic liquid alone at atmospheric conditions. It is also possible to extract lipids from an ionic liquid phase at progressively increasing pressures, and thus obtain fractions that vary in their degree of unsaturation and/or composition in terms of types of lipids. The combined use of ionic liquids with a near-critical fluid to produce fractions rich in 10 unsaturated lipids can be carried out in several different ways. For example, processing can be carried out in a batch-wise, semi-continuous or continuous manner in stirred tank or packed column apparatus. The temperature and pressure conditions under which the near-critical fluid is used can 15 be any suitable temperature and pressure conditions that provide the near-critical -fluid with the desired solvent power. Preferably, the temperatures used are those under which the ionic liquid is in the liquid state. The conditions include, but are not limited to, the reduced temperature (Tr) range [0.75 Tr 1.25] (where Tr is defined as the temperature (T) at which the near-critical fluid is used divided by the critical temperature 20 (T,) of the fluid); and the pressure (P) range [P, P {Pr = 7.00}] (where P, is the fluid vapour pressure at temperature T; and Pr is , the reduced pressure defined as the pressure (P) at which the near-critical fluid is used divided by the critical pressure (Pc) of the fluid). More preferably the pressure is in the range [P, P s {Pr = 4.25}]. For example, suitable conditions for using carbon dioxide (Tc = 304.2 K; Pc = 7.4 MPa) in the 25 present invention include, but are not limited to, the temperature range [273.0 T s 380.3] K, and the pressure range [P, P .51.8] MPa; more preferably the pressure is in the range [P, P 31.5] MPa. In a further example, suitable conditions for using dimethyl ether (Tc = 400.4 K; Pc = 5.4 MPa) in the present invention include, but are not limited to, the temperature range [300.3 s T 500.5] K, and the pressure range [P, P 30 37.8] MPa; more preferably the pressure is in the range P, P 23.0 MPa. In a second aspect the invention provides a process for separating one or more unsaturated lipids from a feed material, comprising at least the steps of: (i) contacting the feed material with a near-critical fluid to give: WO 2009/017425 PCT/NZ2008/000183 17 a. a near-critical solution containing one or more unsaturated lipids; and b. a raffinate; (ii) separating the near-critical solution from the raffinate; 5 (iii) contacting the near-critical solution with an ionic liquid phase to give: c. an ionic liquid phase containing one or more unsaturated lipids; and d. a lipid-depleted near-critical fluid phase; (iv) separating the ionic liquid phase containing one or more unsaturated 10 lipids from the lipid-depleted near-critical fluid phase; and (v) recovering the one or more unsaturated lipids from the ionic liquid phase containing one or more unsaturated lipids. The near-critical solution containing one or more unsaturated lipids can be contacted 15 with the ionic liquid phase in step (iii) of the second aspect of the invention using counter-current or co-current flows in suitable apparatus such as, but not limited to, a packed column, or by passing the near-critical solution into or through ionic liquid. In certain embodiments, recovering the one or more unsaturated lipids according to step 20 (iii) of the first aspect or step (v) of the second aspect of the invention-comprises: (a) contacting the ionic liquid phase containing one or more unsaturated lipids with a near-critical fluid to extract the one or more unsaturated lipids from the ionic liquid phase to give a near-critical solution of one or more unsaturated lipids; and 25 (b) recovering the one or more unsaturated lipids from the near-critical solution by pressure reduction and/or temperature change to reduce the solvent power of the near-critical fluid. Where the ionic liquid phase is a near-critical fluid-containing ionic liquid phase, the 30 pressure of the near-critical fluid can be increased to recover the unsaturated lipids from the ionic liquid phase in step (iii) of the first aspect of the invention. The pressure can be increased in incremental steps to provide fractions that vary in their degree of unsaturation and/or composition in terms of lipid types. Alternatively, a different near critical fluid to that which forms part of the near-critical fluid-containing ionic liquid phase 35 can be used at constant pressure or with incremental pressure steps to recover the WO 2009/017425 PCT/NZ2008/000183 18 unsaturated lipids from the ionic liquid phase in step (iii) of the first aspect of the invention. Advantageously, the temperature and pressure conditions under which the near-critical 5 fluid is used can be varied in order, for example, to remove contaminants, e.g. cholesterol, in step (iii) of the first aspect or step (v) of the second aspect of the invention. In each embodiment the ionic liquid phase can be recovered and reused for subsequent 10 extraction procedures. In some embodiments of the present invention, the ionic liquid phase can be an ionic liquid supported on a suitable solid material and isolation of unsaturated lipids can be achieved using well known chromatographic methodologies. Suitable solid support 15 materials include, but are not limited to, silicas, clays, aluminas, aluminosilicates, other suitable metal oxides and suitable polymeric materials. The solid support materials can be microporous, mesoporous and/or macroporous. Contacting the feed material with the solid-supported ionic liquids can be carried out 20 using any suitable apparatus or methods as are well known in the art. The preferred methods and apparatus include, but are not limited to, contacting in a stirred tank and contacting in a packed column. The recovery of the unsaturated lipids from the solid-supported ionic liquid may be 25 performed by any suitable means. The preferred recovery method is the use of near critical fluids. Mechanism of Action Without wishing to be bound by theory, it is hypothesised that, by virtue of the interaction 30 between the delocalised positive charge on the cation and sites of unsaturation, the ionic liquid forms weak n-bond complexes with unsaturated lipids. The more double bonds present, the stronger the complex. The unsaturated lipids are therefore preferentially dissolved in the ionic liquid phase and are extracted from the lipid containing feed. For example, the invention enables the preferential extraction of 35 unsaturated fatty acids from hydrolysed fish oil (free fatty acids) and from a mixture of WO 2009/017425 PCT/NZ2008/000183 19 fatty acid ethyl esters. In both cases, the PUFA content is preferentially concentrated in the ionic liquid phase with enrichment of SFA and MUFA in the raffinate oil phase. The favourability for ionic liquid-polyunsaturated lipid complex formation can be 5 enhanced using ionic liquids containing anions with some degree of steric shielding around the charge centre. Shielding of the negative charge on the anion reduces the interaction between the cation and anion in the ionic liquid, which enhances the interaction of the cation with the delocalised electrons on the carbon-carbon double bonds in the species of interest, i.e. polyunsaturated lipids. This increased interaction 10 with the polyunsaturated lipid double bonds favours complex formation between the ionic liquid and polyunsaturated lipid thus facilitating separation from species having a lower degree of unsaturation. Steric shielding is brought about by attaching sizeable groups to the anion. This prevents the anion from approaching the cation too closely and reduces the interaction of the anion with the cation positive charge. 15 ABBREVIATIONS PUFA: polyunsaturated fatty acids MUFA: monounsaturated fatty acids 20 SFA: saturated fatty acids EPA: eicosapentaenoic acid DHA: docosahexaenoic acid OA: oleic acid LA: linoleic acid 25 GLA: gamma linoleic acid TAG: triglycerides FAEE: fatty acid ethyl esters GC: gas chromatography. 30 EXAMPLES General The following examples demonstrate the extraction of polyunsaturated lipids from several lipid-containing feed materials using ionic liquids. The ionic liquids used are 35 given in Table 1. The ionic liquids used in the examples are commercially available. Alternatively, ionic liquids may be synthesised by methods well known to those skilled in WO 2009/017425 PCT/NZ2008/000183 20 the art (example syntheses are given in [18]). In some examples liquid solvents are used to recover the dissolved lipophilic compounds from the ionic liquid phase and to recover ionic liquid from the lipid-containing oil phase. In others, near-critical fluids are used to recover the dissolved lipophilic compounds from the ionic liquid phase and regenerate 5 the ionic liquid. Some feed material and ionic liquid solvent miscibilities are given in Table 2 and the appropriate organic wash solvent for each phase is selected from these. Table I - Ionic liquids used in the examples Chemical name Abbreviation 1-ethyl-3-meth'ylimidazolium dicyanamide [emim][dca] 1-butyl-3-methylimidazolium dicyanamide [bmim][dca] 1-butyl-3-methylimidazoilium trifluoromethanesulfonate [bmim][TfO] 1 -butyl-3-methylimidazoilium hexafluorophosphate [bmim][PF 6 ] 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [bmim][BTA] 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [hmim][BTA] 1-ethyl-3-methylpyridinium bis(trifluoromethylsulfonyl)imide [empy][BTA] 10 Table 2 - Solvent miscibility with feed materials and ionic liquids* Hoki Cod Borage Krill femim] [bmim] [bmim] [bmim] [bmim] [hmim] [empy] oil liver oil oil [dca] [dca] [TfO] [PF 6 ] [BTA] [BTA] [BTA] oil Water X X X X 0 0 0 X X X X Hexane 0 0 0 0 X X X - X X X Ethanol - - - - - 0 - - 0 0 0 Chloroform 0 0 0 - - 0 0 - 0 0 Toluene 0 0 0 0 X X X X X X X Ethyl 0 0 0 - X - 0 0 0 0 0 acetate 2-Propanol - - - - - 0 - - 0 0 0 2-Butanol - - - - - 0 - - 0 0 0 Petroleum 0 0 0 0 - X - - X X X ether Diethyl 0 0 0 - X X X X X X X ether *0 = miscible, X = immiscible, - = not tested 15 Example 1 - PUFA from hoki liver oil This example shows that polyunsaturated fatty acids (PUFA) can be concentrated from lipid-containing feed materials using ionic liquids. The PUFA, in the form of free fatty acids, are extracted from hydrolysed liver oil from the hoki fish. 15-20 g of ionic liquid and around 10 g of hydrolysed hoki liver oil are placed in a 150 mL glass vessel. The WO 2009/017425 PCT/NZ2008/000183 21 150 mL vessel is placed inside a jacketed glass water bath which is maintained at 313 K using a reticulated water supply. The mixture is stirred for 4 hours using a cross-shaped magnetic stirrer bead to provide sufficient mixing. The mixture is then transferred into a separating funnel and allowed to partition. The lower ionic liquid phase is removed and 5 the upper hoki oil phase is retained. Each phase is washed with the appropriate solvent(s) (see Table 2) to recover the lipids/ionic liquids and the lipid samples are analysed by GC. The PUFA are concentrated in the lower ionic liquid phase and the saturated fatty acids (SFA) and mono-unsaturated fatty acids (MUFA) are concentrated in the upper liquid oil phase. This is shown by the PUFA/SFA ratio given in Table 3. 10 Compared to the feed material, the PUFA/SFA ratio decreases for the residual oil layer and increases for the ionic liquid oil. The fatty acid profiles of the hydrolysed hoki oil feed, the upper oil phase (residual oil after extraction), and the oil recovered from the ionic liquid (ionic liquid oil) are given in Table 3. The fatty acids are designated as follows: X:Y w-z, where X is the number of carbon atoms in the fatty acid, Y is the 15 number of double bonds, and z is the position of the first double bond. The enhancement of the PUFA, omega-3 (w3) PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations in the oil recovered from the ionic liquid, over the concentrations in the feed, are shown. Of the three ionic liquids tested in this example [emim][dca] produced the most PUFA-rich oil fraction, with the ionic liquid oil 20 showing an almost twofold increase in total PUFA when compared to the concentration in the hoki oil feed. Table 3 - Fatty acid profiles of the hoki oil feed, the upper oil phase (residual oil) and the oil recovered from the ionic liquid phase (ionic liquid oil) [emim][dca] [bmim][TfO] [bmim][PFs] Fatty acid Feed Residual Ionic Residual Ionic Residual Ionic oil liquid oil oil liquid oil oil liquid oil Weight % of total fatty acids 14:0 3.6 3.6 3.7 3.3 4.6 3.7 3.5 16:0 15.9 14.6 10.2 15.8 13.5 15.4 13.5 16:1 w7 5.6 6.4 6.4 5.9 7.4 6.3 6.2 17:0 1.3 1.1 1.3 1.0 1.3 1.1 1.2 16:3 0.7 1.4 1.1 18:0 2.8 4.9 2.6 5.5 3.1 4.9 4.3 18:1W9 23.7 25.6 16.9 25.8 20.5 24.3 23.0 18:1 w7 3.2 3.7 2.4 3.7 3.0 3.4 3.3 18:2 w6 2.7 3.6 3.4 3.2 3.7 3.3 3.6 18:3 w3 0.6 0.9 1.4 0.8 1.2 0.9 1.1 WO 2009/017425 PCT/NZ2008/000183 22 18:4 w3 0.8 1.1 3.0 0.8 1.9 1.1 1.7 20:1 w9 8.9 6.6 3.4 7.2 3.8 6.2 5.5 20:4 w6 0.7 1.1 1.0 20:4 w3 1.4 1.4 1.8 1.1 1.6 1.2 1.5 EPA 20:5 w3 5.5 6.2 14.6 5.4 9.7 6.4 9.0 22:1 wl1 3.3 2.1 0.9 2.5 0.9 2.0 1.7 22:5 w3 2.0- 2.7 3.5 2.5 3.0 2.6 3.0 DHA 22:6 w3 12.1 9.8 18.4 8.8 14.2 9.9 13.0 24:1 1.5 0.9 1.3 1.0 Total SFA 23.5 24.3 17.9 25.7 22.5 25.2 22.6 Total MUFA 46.2 45.2 29.9 46.4 35.6 43.3 39.6 Total PUFA 26.4 25.7 50.0 22.6 37.4 25.4 32.9 Total w3 22.4 22.1 42.7 19.4 31.6 22.0 29.4 PUFA PUFA/SFA 0.95 0.91 2.39 0.75 1.40 0.87 1.30 ratio Example 2 - PUFA from borage oil This example shows that PUFA can be further concentrated from a lipid-containing feed material which is already rich in PUFA. Hydrolysed borage oil (fatty acids in the form of 5 free fatty acids) is pre-treated with urea to enhance the oil's PUFA content, and the resulting PUFA-rich oil is then extracted with ionic liquids. The methodology for the extraction of PUFA from the PUFA-rich borage oil is the same as that described for hydrolysed hoki liver oil in Example 1. The results are given in Table 4, which shows the concentrations of oleic acid (OA), linoleic acid (LA), garnma-linoleic acid (GLA) and total 10 PUFA in the PUFA-rich borage oil feed, the upper oil phase (residual oil after extraction) and the oil recovered from the ionic liquid (ionic liquid oil). Here, the ionic liquids [bmim][BTA] and [empy][BTA] are the most effective in further concentrating GLA, from 44.5 % in the feed to 54 % in the extract. 15 Table 4 - Concentrations of oleic acid (OA), linoleic acid (LA), gamma-linoleic acid (GLA) and total PUFA in the borage oil feed, the upper oil phase (residual oil) and the oil recovered from the ionic liquid (ionic liquid oil) Oil OA LA GLA PUFA Ionic liquid Weight % of total fatty acids Feed 2.4 53.1 44.5 97.6 [bmim][BTA] Residual oil 2.4 53.3 44.3 97.6 Ionic liquid oil 45.8 54.2 100 [hmim][BTA] Residual oil 2.6 53.3 44.1 97.4 WO 2009/017425 PCT/NZ2008/000183 23 Ionic liquid oil 1.8 48.8 49.4 98.2 Residual oil 2.6 53.2 44.3 97.5 Ionic liquid oil 1.4 44.2 54.5 98.7 Example 3 - PUFA from hoki fish oil FAEE In this example PUFA are extracted from a mixture of fatty acid ethyl esters (FAEE) derived from hoki fish oil. The methodology is the same as that described in Example 1. 5 The total SFA, MUFA and PUFA (with particular reference to EPA and DHA) concentrations of the feed oil, upper oil phase (residual oil after extraction) and oil recovered from the ionic liquid (ionic liquid oil) are given in Table 5 along with the PUFA/SFA ratio. Compared to the feed, the PUFA/SFA ratio decreases for the residual oil layer and increases for the ionic liquid oil, except for the ionic liquid oil from extraction 10 with [hmim][BTA], where the enhanced level of PUFA is mainly attributed to reduced levels of MUFA in the extract. Table 5 - Total SFA, MUFA, EPA, DHA and PUFA concentrations of the feed oil, upper oil phase (residual oil) and oil recovered from the ionic liquid (ionic liquid 15 oil) SFA MUFA EPA ] DHA 1 PUFA JPUFA/SFA Oil ratio Ionic liquid Weight % of total fatty acids Feed 20.6 43.6 6.8 18.7 34.7 1.68 [bmim][dca] Residual oil 21.6 43.5 6.4 17.5 32.9 1.52 l Ionic liquid oil 18.4 32.9 10.8 24.4 44.3 2.41 [bmim][BTA] Residual oil 21.9 43.8 6.4 17.5 32.2 1.47 Ionic liquid oil 21.1 31.6 11.1 22.3 45.5 2.16 [hmim][BTA] Residual oil 22.4 43.7 6.0 16.8 31.3 1.40 Ionic liquid oil 22.5 37.4 8.5 18.9 37.9 1.68 Residual oil 22.4 43.1 6.2 16.8 31.8 1.42 Ionic liquid oil 19.7 33.2 10.8 23.3 45.5 2.31 Example 4 - EPA and DHA enrichment of Hoki fish oil FAEE This example shows that ionic liquids can be used to further enrich the EPA and DHA content of lipid-containing feed materials which already contain enhanced levels of these 20 fatty acids. A mixture of FAEE derived from hoki oil is subjected to a molecular distillation process to produce a mixture of FAEE with an enhanced concentration of long-chain fatty acids (including EPA and DHA). Ionic liquids are used to extract PUFA from the long-chain fatty acid-rich FAEE mixture using the methodology described in Example 1. The total SFA, MUFA and PUFA (with particular reference to EPA and DHA) WO 2009/017425 PCT/NZ2008/000183 24 concentrations of the feed oil, upper oil phase (residual oil after extraction) and oil recovered from the ionic liquid (ionic liquid oil) are given in Table 6 along with the PUFA/SFA ratio. Compared to the feed, the PUFA/SFA ratio decreases for the residual oil layer and increases for the ionic liquid oil. The ionic liquid [bmim][PF 6 ] is particularly 5 selective for the extraction of PUFA over the less saturated FAEE from this feed material. Table 6 - Total SFA, MUFA, EPA, DHA and PUFA concentrations of the feed oil, upper oil phase (residual oil) and oil recovered from the ionic liquid (ionic liquid 10 oil) SFA MUFA EPA DHA PUFA PUFA/SFA Oil ratio Ionic liquid Weight % of total fatty acids Feed 4.2 44.8 9.0 26.7 43.8 10.4 [emim][dca] Residual oil 4.2 45.0 8.9 26.7 .42.3 10.1 l Ionic liquid oil 4.0 42.4 10.1 28.8 45.5 11.4 Residual oil 4.1 45.0 8.5 26.3 41.5 10.1 [bmimllTfO] Ionic liquid oil 4.1 41.3 10.8 29.4 47.1 11.5 Residual oil 4.1 44.9 8.8 26.8 42.2 10.3 Ionic liquid oil 2.4 23.7 17.7 40.9 68.7 28.6 Example 5 - astaxanthin from kril oil This example shows that lipid polyunsaturates aside from fatty acids and their 15 derivatives can be extracted using ionic liquids. In this example, astaxanthin and astaxanthin esters present in oil extracted from krill powder can be enriched using ionic liquids. The remainder of the oil is TAG with low levels of PUFA. The. methodology is the same as that used in Example 1 and the results are given in Table 8. [bmim][PF 6 ] is particularly effective in this example, increasing the concentration in the extract by a 20 factor of 30. Oa t HO O astaxanthin WO 2009/017425 PCT/NZ2008/000183 25 Table 7 - Enrichment of astaxanthin from krill oil Ionic liquid Oil Weight % of astaxanthin Feed 0.012 . Residual oil 0.011 -T Ionic liquid oil 0.036 .P Residual oil 0.007 [bm P6 Ionic liquid oil 0.368 5 Example 6 - Concentration of PUFA using [bmim][PFJ and supercritical carbon dioxide This example shows that concentration of PUFA can be achieved from an ionic liquid/near-critical fluid mixture, and that fractionation of lipids can be achieved by 10 varying the extraction pressure. The example also shows that fatty acid ethyl esters (FAEE) can be separated from non FAEE using ionic liquid/near-critical fluid mixtures. In this example PUFA are extracted from a PUFA-rich mixture of fatty acid ethyl esters (FAEE) derived from hoki oil using [bmim][PF] and supercritical carbon dioxide. The PUFA-rich mixture of FAEE is obtained by combining FAEE from the urea fractionation 15 of Hoki oil [19], containing approximately 70 % PUFA,, with FAEE obtained from the enzymatic ethanolysis of hoki oil (e.g. by the process given in [20]), containing approximately 10 % PUFA, in proportions giving the composition reported in table 8. The FAEE from the enzymatic ethanolysis of hoki oil also contained low levels of cholesterol, monoglycerides and triglycerides. 100 g of [bmim][PF 6 ] and 50 g of esterified hoki liver 20 oil are loaded into a 300 mL high-pressure vessel, to which CO 2 is then added. The vessel is maintained at 313 K using a water bath. The ionic liquid/FAEE oil mixture is stirred under a constant CO 2 pressure of 8.0 MPa for 2 hours using an overhead stirrer to ensure thorough mixing. After 2 hours the mixture is continuously stirred and extracted with supercritical CO 2 using incremental pressure steps from 8.0 to 25.0 MPa. 25 The CO 2 is continuously passed through the ionic liquid/FAEE oil mixture using a dip tube inserted into the liquid phase. There is no equilibration period between pressure increases and extractions at a given pressure are carried out for 15 minutes. Extraction samples are collected in a separator by pressure reduction to approximately 4.8 MPa

(CO

2 cylinder pressure). After extraction at the highest pressure the stirring is stopped 30 and the residual mixture in the high-pressure vessel is allowed to settle. After extraction at 15.0 MPa the residual ionic liquid is withdrawn from the bottom of high-pressure vessel under 15.0 MPa pressure. After reduction to atmospheric pressure, the ionic WO 2009/017425 PCT/NZ2008/000183 26 liquid layer sample splits into two phases: i) a lower lipid-containing ionic liquid-rich phase and, ii) an upper oil-rich phase. The phase separation of the ionic liquid layer at atmospheric pressure indicates that dissolution of CO 2 in the ionic liquid makes the ionic liquid more lipophilic, thus increasing the solubility of the FAEE oil in the ionic liquid over 5 that at atmospheric conditions. In addition the phase separation suggests that extraction at 15.0 MPa is not sufficient to effectively regenerate [bmim][PF 6 ] for further use after processing FAEE. The lipid compositions of the separator samples are analysed by GC and thin-layer chromatography (TLC). The total SFA, MUFA and PUFA (with particular reference to EPA and DHA) concentrations of the separator samples are given in Table 10 8 along with the PUFA/SFA ratio. Some degree of fractionation is achieved during extraction with C0 2 , which is indicated by the variation in composition of the separator samples and variation in the PUFA/SFA ratios at different pressures. The bulk of the lipid is extracted at 12.0 MPa. The extract at 15.0 MPa is enriched in non-FAEE components, particularly TAG, which had low levels of PUFA. 15 Table 8 - Total SFA, MUFA, EPA, DHA and PUFA concentrations of the separator samples after extraction from [bmim][PF6] with CO 2 at 8.0 to 15.0 MPa Extraction SFA MUFA EPA DHA PUFA PUFA/SFA % total CO 2 pressure Weight % of total fatty acids ratio oil loading /MPa extracted /g oil Feed 16.4 38.1 7.7 15.8 35.1 2.1 per kg

CO

2 8.0 13.5 31.2 1.9 2.1 26.6 2.1 0.89 0.4 12.0 14.8 37.4 7.7 17.5 37.2 2.5 92.70 107.8 15.0 21.1 39.1 6.2 13.8 28.8 1.4 6.41 5.9 Example 7 - Concentration of PUFA using ionic liquids and supercritical fluids 20 This example shows that concentration of PUFA can be achieved from a range of ionic liquid/near-critical fluid mixtures, and that fractionation of lipids can be achieved by varying the extraction pressure. The example also shows that fatty acid ethyl esters (FAEE) can be separated from non FAEE using ionic liquid/near-critical fluid mixtures; the ionic liquid can be completely regenerated using the near-critical fluid; and the ionic 25 liquid can be re-used with no loss of efficiency. In this example PUFA are extracted from a PUFA-rich mixture of fatty acid ethyl esters (FAEE) derived from hoki oil and prepared in a similar manner to Example 6 using ionic liquids and supercritical carbon dioxide. The methodology is similar to that used in Example 6 except that 150 g of ionic WO 2009/017425 PCT/NZ2008/000183 27 liquid/FAEE oil mixtures of varying mass ratios (see Tables 9 and 10) are placed into the 300 mL high-pressure vessel, and a pressure of 8.5 MPa is used during the initial 2 hour equilibration period. The incremental pressure steps ranged from 8.5 to 25.0 MPa with a 15 minute equilibration period at a given pressure before extraction at that pressure for 5 15 minutes. The [bmim][PF 6 ] used in Example 6 is regenerated using toluene and toluene/hexane mixtures and dried to constant weight using rotary evaporation. Regenerated [bmim][PF 6 ] is used in this Example. After extraction at the highest pressure, the residual ionic liquids are withdrawn from the bottom of high-pressure vessel under that pressure. Upon reduction to atmospheric pressure, no obvious phase 10 separation is observed. The recovered ionic liquids are washed with the appropriate solvent(s) from Table 2 and the wash contained no traceable quantities of lipid (as shown by TLC and GC of the solvent washes), showing that extraction with CO 2 at high pressures is an effective method of ionic liquid regeneration. The same batch of [bmim][BTA] is used for three consecutive experiments in this example - the 15 [bmim][BTA] is regenerated by extraction with CO 2 at 25.0 MPa. The three experiments with the same batch of [bmim][BTA] gave comparable results demonstrating that ionic liquids can be re-used after regeneration by supercritical fluid extraction. The total SFA, MUFA and PUFA (with particular reference to EPA and DHA) concentrations of the separator samples are given in Table 9 and 10 along with the PUFA/SFA ratio. Some 20 degree of fractionation is achieved during extraction with CO 2 , which is indicated by the variation in composition of the separator samples and variation in the PUFA/SFA ratios at different pressures. The final fraction obtained at 25.0 MPa is usually highly coloured. TLC shows that this final fraction contains only low levels of FAEE, and high levels of cholesterol and TAG. The TAG have low levels of PUFA, which is why the final PUFA 25 content is lower than the fractions obtained at 15.0 and 20.0 MPa. Two different feeds are used, the compositions of which are given in Tables 9 and 10. Table 9 - Total SFA, MUFA, EPA,. DHA and PUFA concentrations of the separator samples after extraction from different ionic liquids with CO 2 at 8.0 to 25.0 MPa Extraction SFA MUFA EPA DHA PUFA PUFA/SFA % total CO 2 pressure Weight % of total fatty acids ratio oil loading IMPa extracted /g oil Feed 16.4 38.1 7.7 15.8 35.1 2.1 per kg

CO

2 Regenerated [bmim][PF 6 ], 2: 1, ionic liquid oil ratio 8.5 23.7 38.8 6.0 9.4 26.2 1.1 4.57 1.9 WO 2009/017425 PCT/NZ2008/000183 28 12.0 17.9 38.6 7.5 13.8 33.1 1.8 55.34 24.6 15.0 12.8 37.4 8.2 19.8 40.2 3.1 29.82 16.0 20.0 15.6 34.5 7.3 20.5 38.2 2.4 5.51 4.4 25.0 21.0 35.1 6.7 15.8 31.5 1.5 4.77 2.6 [emim][dca], 2: 1, ionic liquid: oil ratio 8.5 23.6 37.6 6.1 10.2 26.6 1.1 2.59 1.5 12.0 18.4 36.9 6.8 11.7 32.0 1.7 19.00 23.7 :15.0 16.5 38.8 7.6 14.6 34.1 2.1 24.26 78.3 20.0 14.3 38.2 7.9 17.8 37.5 2.6 48.02 22.3 25.0 20.6 39.7 6.5 14.1 29.8 1.4 6.14 3.4 [bmim][BTA], 2: 1, ionic liquid : oil ratio 8.5 25.3 38.3 5.8 9.4 25.5 1.0 2.86 1.3 12.0 19.2 38.9 7.2 12.5 31.1 1.6 34.27 36.4 15.0 14.6 38.5 8.0 16.8 36.9 2.5 50.03 40.8 20.0 12.5 35.9 8.1 21.2 41.0 3.3 12.84 14.2 Table 10 - Total SFA, MUFA, EPA, DHA and PUFA concentrations of the separator samples after extraction from [bmim][BTA] with varying ionic liquid:oil ratios with 5 CO 2 at 8.0 to 25.0 MPa Extraction SFA MUFA EPA DHA PUFA PUFAISFA % total CO 2 pressure Weight % of total fatty acids ratio oil loading /MPa extracted /g oil Feed 15.5 35.5 8.4 17.4 38.2 2.5 per kg Co 2 [bmim][BTA], 3: 1, ionic liquid : oil ratio 8.5 25.3 38.6 6.1 8.7 25.5 1.0 2.74 1.1 12.0 16.6 36.4 8.1 15.0 35.6 2.1 51.61 32.2 15.0 11.9 34.2 9.0 21.4 43.4 3.6 35.30 19.0 20.0 15.1 33.7 8.1 20.3 39.2 2.6 5.44 3.0 25.0 21.2 36.3 7.0 14.9 31.1 1.5 4.91 2.0 Regenerated [bmim][BTA], 3: 1, ionic liquid : oil ratio 8.5 23.7 36.9 6.3 10.0 26.9 1.1 1.70 1.5 12.0 16.9 36.5 8.1 14.9 35.6 2.1 53.11 38.0 15.0 11.9 34.2 9.0 21.4 43.5 3.7 33.59 25.4 20.0 12.8 31.1 7.9 20.2 41.3 3.2 6.60 6.4 WO 2009/017425 PCT/NZ2008/000183 29 25.0 19.4 33.2 7.0 14.9 33.5 1.7 5.01 3.1 2" Regeneration of [bmim][BTA], 4: 1, ionic liquid: oil ratio 8.5 20.6 36.1 7.3 14.8 32.7 1.6 1.03 0.8 12.0 17.7 36.8 8.1 14.6 35.1 2.0 51.31 32.4 15.0 11.9 34.6 9.1 21.4 43.6 3.7 35.36 22.9 20.0 14.0 32.9 8.4 21.7 41.3 3.0 7.20 4.8 25.0 20.6 35.1 7.3 15.7 32.6 1.6 5.10 2.2 Example 8 - Reduction of non-PUFA contaminants from a PUFA concentrate using ionic liquids and supercritical fluids This example shows that the level of non-PUFA contaminants from a PUFA-rich fatty 5 acid ethyl ester (FAEE) concentrate can be reduced using an ionic liquid/near-critical fluid mixture, and that fractionation of lipids can be achieved by varying the extraction pressure. The ionic liquid [bmim][BTA] is used. The PUFA-rich FAEE concentrate feed is obtained from the urea fractionation of Hoki oil [19] and the feed material composition is given in Table 11. The methodology is the same as that used in Example 7 except an 10 initial pressure of 9.0 MPa is used during the equilibration stage. The ionic liquid to fatty acid ethyl ester ratio is 2:1 by mass with a total of 150g of ionic liquid loaded into the high-pressure vessel. After extraction at 25.0 MPa the residual -ionic liquid is withdrawn from the bottom of high-pressure vessel under 25.0 MPa pressure. Upon reduction to atmospheric pressure, no obvious phase separation is observed. The recovered 15 [bmim][BTA] is washed with the appropriate solvent(s) from Table 2 and the wash contained no traceable quantities of lipid (as shown by TLC and GC of the solvent washes), showing that extraction with CO 2 at high pressures is an effective method of ionic liquid regeneration. This example also shows that fatty acid ethyl esters (FAEE) can be separated from non FAEE using ionic liquid/near-critical fluid mixtures. TLC of 20 the final fraction obtained at 25.0 MPa showed reduced levels of FAEE, and high levels of the non-PUFA contaminants cholesterol and free fatty acids. The total SFA, MUFA and PUFA (with particular reference to EPA and DHA) concentrations of the separator samples are given in Table 11 along with the PUFA/SFA ratio. Some degree of fractionation is achieved during extraction with C0 2 , which is indicated by the variation in 25 composition of the separator samples and variation in the PUFA/SFA ratios at different pressures. The fraction obtained at 9.0 MPa is enriched in non-PUFA contaminants, particularly low molecular weight fatty acid ethyl esters C16:1 and C18:1.

WO 2009/017425 PCT/NZ2008/000183 30 Table 11 - Total SFA, MUFA, EPA, DHA and PUFA concentrations of the separator samples after extraction from [bmim][BTA] with CO 2 at 8.0 to 25.0 MPa Extraction SFA MUFA EPA DHA PUFA PUFAISFA % total CO 2 pressure Weight % of total fatty acids ratio oil loading /MPa extracted Ig oil Feed 1.2 15.3 14.8 35.3 70.2 58.5 per kg

CO

2 9.0 2.9 22.5 14.0 23.4 58.0 20.0 5.78 5.3 12.0 0.8 17.0 15.1 31.4 67.4 84.3 39.34 38.5 15.0 0.5 12.6 14.8 39.7 74.1 148.2 45.32 38.7 20.0 1.5 14.0 13.4 40.7 70.8 47.2 7.35 6.8 25.0 5.8 23.8 11.1 32.0 56.8 9.8 2.21 1.3 5 Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in the specification. 10 INDUSTRIAL APPLICABILITY The process of the invention relates to the use of an ionic liquid for extracting unsaturated lipophilic compounds from lipid containing feed materials. The process is particularly useful for extracting polyunsaturated fatty .acids, or their derivatives, and polyunsaturated carotenoids, from feed materials. 15 WO 2009/017425 PCT/NZ2008/000183 31 REFERENCES [1] Shahidi, F. "Marine Nutraceuticals" Inform, 13, 2002, 57-62. [2] Stansby, M. E. "Nutritional Properties of Fish Oil for Human Consumption Modem Aspects" in Chapter 2 of Fish Oils in Nutrition, ed. M. E. Stansby, Van 5 Nostrand Reinhold, New York, 1990. [3] Stout, V. F.; Nilsson, W. B.; Krzynowek, J. and Schlenk, H. "Fractionation of Fish Oils and Their Fatty Acids" in Chapter 4 of Fish Oils in Nutrition, ed M. E. Stansby, Van Nostrand Reinhold, New York, 1990. [4] Bengen, M. F., Patent in German "Verfahren zur Zerlegung von Gemischen 10 organischer Verbindungen", DE 869070, 1953. [5] Seike, Y.; Kamio, E.; Ono, T.; Yoshizawa, H. "Extraction of Ethyl Ester of Polyunsaturated Fatty Acids by Utilizing Slug Flow Prepared by Microreactor" J. Chem. Eng. Japan. 40, 2007, 1076-1084. [6] Beebe, L. M.; Brown, P. R.; Turcotte, L. G.; "Preparative-Scale-High 15 Performance Liquid Chromatography of Omega-3 Polyunsaturated Fatty Acid Esters Derived from Fish Oil, J. Chromatogr. 495, 1988, 369. [7] Adlof, R. 0.; Emiken, E. A.; "The Isolation of Omega-3 Polyunsaturated Fatty Acids and Methyl Esters of Fish Oils by Silver Resin Chromatography" J. Am. Oil Chem. Soc. 62, 1985,1592. 20 [8] Nillson, W. B.; Gauglitz, E. J.; Hudson, J. K.; Stout, V. F.; Spinelli, J. "Fractionation of Menhaden Oil Ethyl Esters using Supercritical Fluid C0 2 " J. Am. Oil Chem. Soc. 65,1988,109-117. [91 Nillson, W. B.; Gauglitz, E. J.; Hudson, J. K. "Supercritical Fluid Fractionation of Fish Oil Ethyl Esters using Incremental Pressure Programming and a 25 Temperature Gradient" J. Am. Oil Chem. Soc. 66, 1989, 1596-1600. [10] Catchpole, 0. J., Grey, J. B.; Noermark, K. A. "Fractionation of Fish Oils using Supercritical CO 2 and CO 2 + Ethanol Mixtures" J. Supercrit. Fluids, 19, 2000, 25 37. [11] Anderson, J. L.; Armstrong, D. W. "Immobilized Ionic Liquids as High 30 Temperature/High-Selectivity/High-Stability Gas Chromatography Stationary Phases", Anal. Chem. 77, 2005, 6453-6462. [12] Li, M.; Li, T. "Enrichment of Omega-3 Polyunsaturated Fatty Acid Methyl Esters by Ionic Liquids Containing Silver Salts" J. Sep. Sci. Technol. 43, 2008, 2072 2089. 35 [13] Seddon, K. R. "Ionic Liquids for Clean Technology" J. Chem. Tech. Biotechnol. 68,1997, 351-356.

WO 2009/017425 PCT/NZ2008/000183 32 [14] Enderby, J. E. "lonic Liquids: Recent Progress and Remaining Problems" J. Phys. Condensed Matter, 5 (Supp. 34), 1993, B99-B106. [15] Freemantle, M. "Designer Solvents" Chemical and Engineering News, 1998, March 30, 32-37. 5 [16] Gordon, C. M.; Holbrey, J. D.; Kennedy, A. R.; Seddon, K. R. "Ionic Liquid Crystals: Hexafluorophosphate Salts" J. Mater. Chem. 8, 1998, 2627-2636. [17] Welton, T. "Room-Temperature Ionic Liquid Solvents for Synthesis and Catalysis" Chem. Rev. 99,1999, 2071-2084. [18] Rogers, R. D.; Seddon, K. R. (Eds.) "Ionic Liquids as Green Solvents: Progress .10 and Prospects" ACS Symposium Series 856, American Chemical Society, Washington DC, 2003, ppl4-107. [19] Catchpole, O.J., MacKenzie, A.D. and Grey, J.B., Patent WO 03/089399, 2003. [20] Weber, A.; Catchpole, 0. J.; Eltringham, W. "Supercritical fluid assisted, integrated process for the synthesis and separation of different lipid derivatives" 15 J. Sep. Sci. 31, 2008, 1346-1351.

Claims (18)

1. A process for separating one or more unsaturated lipids from a feed material, comprising at least the steps of: 5 (i) contacting the feed material with an ionic liquid phase to give: a. an ionic liquid phase containing one or more unsaturated lipids; and b. a raffinate; (ii) separating the ionic liquid phase containing one or more unsaturated 10 fipids from the raffinate; and (iii) recovering one or more unsaturated lipids from the ionic liquid phase containing one or more unsaturated lipids.

2. A process for separating one or more unsaturated lipids from a feed material, 15 comprising at least the steps of: (i) contacting the feed material with a near-critical fluid to give: a. a near-critical solution containing one or more unsaturated lipids; and b. a raffinate; 20 (ii) separating the near-critical solution from the raffinate; (iii) contacting the near-critical solution with an ionic liquid phase to give: c. an ionic liquid phase containing one or more unsaturated lipids; and d. a lipid-depleted near-critical fluid phase. 25 (iv) separating the ionic liquid phase containing one or more unsaturated lipids from the lipid-depleted near-critical fluid phase; and (v) recovering the one or more unsaturated lipids from the ionic liquid phase containing one or more unsaturated lipids. 30

3. A process as claimed in claim 1 or claim 2 where the raffinate is a liquid.

4 A process as claimed in any one of claims 1 to 3 where any one or more of the unsaturated lipids are polyunsaturated fatty acids (PUFA) or fatty acid derivatives. WO 2009/017425 PCT/NZ2008/000183 34

5. A process as claimed in claim 4 where the fatty acid derivatives are selected from the group comprising fatty acid C-C 4 alcohol esters, fatty acid amines, fatty acid ethanolamines, fatty acid amides, and fatty alcohols. 5

6. A process as claimed in claim 4 or claim 5 where the PUFA or fatty acid derivatives are selected from cis-5,8,11,14,17-eicosapentaenoic acid, cis 4,7,10,13,16,19-docosahexaenoic acid, 6,9,12-octadecatrienoic acid (y-linolenic acid), 9,12,15-octadecatrienoic acid (a-linolenic acid), 9,11-octadecadienoic acid (conjugated linoleic acid), and esters thereof. 10

7. A process as claimed in any one of claims 1 to 3 where the one or more unsaturated lipids are selected from the group comprising carotenoids, partial glycerides, triglycerides, hydrocarbons, alkyldiacyl glycerides, fat soluble vitamins, tocotrienols, phospholipids, glycolipids, and sphingolipids. 15

8. A process as claimed in any one of claims 1 to 7 where the ionic liquid comprises a cation selected from the group comprising substituted-quinolinium [R 1 .10Chin]*, substituted-imidazolium [R 1 .- lM]*, substituted-pyridinium [R 1 .ePy]*, substituted ammonium [Rl4N]*, substituted-phosphonium [R 1 . 4 P]*, substituted-pyrrolidinium 20 [R 1 - 10 Pyrr]*, where each R is hydrogen, alkyl, substituted-alkyl, alkoxy, substituted alkoxy, aryl or substituted-aryl, or combinations thereof.

9. A process as claimed in any one of claims 1 to 8 where the ionic liquid comprises an anion selected from the group comprising acetate [OAc]-, bis[1,2 25 benzenediolato(2-)-OO']borate [BBB]-, tetracyanoborate [B(CN) 4 1-, tetrafluoroborate [BF4]-, bis(methylsulfonyl)imide [BMA]~, bis(malonato(2-))borate [BMB]-, bis(oxalate(2-))borate [BOB]-, bis(salicylato(2-))borate [BSB]-, bis(trifluoromethylsulfonyl)imide [BTA]-, trifluoromethylsulfonate [CF 3 SO 3 ]~, methylsulfonate [CH 3 SO 3 ]-, methylsulfate [CH 3 SO 4 ]~, ethylsulfate [C 2 H 5 SO 4 ]-, 30 propylsulfate [C 3 H 7 SO4]-, butylsulfate [C 4 H 9 SO 4 1, pentylsulfate [C 5 H 11 S0 4 ]-, hexylsulfate [C 6 H 13 SO 4 ]~, heptylsulfate [C 7 H 15 SO 4 ]-, octylsulfate. [CBH 17 SO 4 ]-, chloride [Cl]-, dimethylphosphate [DMPO4]-, hydrogensulfate [HSO 4 ]~, N methylsulfonylacetamide [MAcA]-, 2-(2-methoxyethoxy)ethylsulfate [MDEGSO 4 ]-, dicyanamide [N(CN) 2 1-, hexafluorophosphate [PF 6 ]-, thiocyanate [SCN]~, p 35 toluenesulfonate [TOS]~, and salicylate [Sal]~. WO 2009/017425 PCT/NZ2008/000183 35

10. A process as claimed in any one of claims 1 to 9 where the ionic liquid phase consists of a single ionic liquid or a mixture of ionic liquids, or a mixture of a single ionic liquid and a near-critical fluid, or a mixture of more than one ionic liquid and a near-critical fluid. 5

11 A process as claimed in claim 10 where the pressure of the near-critical fluid is increased to recover the unsaturated lipids from the ionic liquid phase.

12. A process as claimed in claim 10 or claim 11 where the pressure of the near 10 critical fluid'is increased in incremental steps to give one or more lipid-containing fractions, where when more than one fraction is obtained, the fractions do not have the same lipid composition.

13. A process as claimed in any of claims 1 to 9 where the ionic liquid phase is a 15 mixture of an ionic liquid and an organic solvent or a mixture of organic solvents.

14. A process as claimed in any one of claims 1 to 13 where recovering the one or more unsaturated lipids according to step (iii) of claim 1 or step (v) of claim 2 comprises: 20 (a) contacting the ionic liquid phase containing one or more unsaturated lipids with a near-critical fluid, to extract the one or more unsaturated lipids from the ionic liquid phase to give a near-critical solution of one or more unsaturated lipids; and (b) recovering the one or more unsaturated lipids from the near-critical 25 solution by pressure reduction and/or temperature change to reduce the solvent power of the near-critical fluid.

15. A process as claimed in any one of claims 1 or 3 to 13 where contacting the feed material with an ionic liquid phase according to step (i) comprises: 30 (a) contacting an ionic liquid with a near-critical fluid to give a near-critical fluid-containing ionic liquid phase; and (b) contacting the feed material with the near-critical fluid-containing ionic liquid phase to give: i. a near-critical fluid-containing ionic liquid solution phase 35 containing one or more unsaturated lipids; and ii. a raffinate. WO 2009/017425 PCT/NZ2008/000183 36

16. A process as claimed in any one of claims 1 or 3 to 13 where contacting the feed material with an ionic liquid phase according to step (i) comprises: (a) contacting the ionic liquid with the feed material to give: 5 i. an ionic liquid phase containing one or more unsaturated lipids; and ii. a raffinate; and (b) contacting the ionic liquid phase and the raffinate with a near-critical fluid to give: vii. a near-critical fluid-containing ionic liquid solution phase 10 containing one or more unsaturated lipids; and viii. a raffinate.

17. A process as claimed in claim 1 or claim 2 where the ionic liquid phase containing one or more unsaturated lipids is in the form of a solution. 15

18. A process as claimed in claim 1 or claim 2 where the ionic liquid phase is a solid supported ionic liquid and the ionic liquid phase containing one or more unsaturated lipids is in the form of one or more unsaturated lipids adsorbed to the solid-supported ionic liquid.