Synthesis of some deep eutectic solvents from 2 - Alkylbenzimidazole, choline chloride and their application for extraction of omega - 3, 6, 9 from the fat of vietnamese basa fish in Mekong delta

procedure of material processing Raw materials of Vietnamese basa fish collected from seafood processing factories in Dong Thap were washed and drained. Then the fish were cut by following the process by export seafood factories and 3 parts were obtained, including primary fat, meat (fillets) and the rest (skin, head, body, organs ...) which were described as the by-products in the table. We performed three times of fish cutting procedures to determine the average weight. Table 2.2. Components of Vietnamese basa fish at the first stage of process Tra fish Basa fish No Components Weight (gam) Percentage (%) Weight (gam) Percentage (%) 1 Primary fat 50 2,3 106 6,06 2 Meat (phile) 816 37,1 615 35,14 3 By-products 1334 60,6 1029 58,8 Total 2200 100 1750 100 7 2.2.2. The methods of fat extraction from the by-products The by-products were added with water and boiled for 60 minutes, then they were allowed to cool and cooled. The fat obtained from the by-product was separated and put under serial extraction times with the n-hexane-methanol solvent system. Methylation: The jelly obtained after extraction was methylated with methanol and concentrated sulfuaric acid was used as catalyst with the ratio of extracts/Methanol/Catalytic 50gam/ 100gam/1gam. The esterification was carried out in a 200 ml glass flask, with reflux condenser attached for 3 hours under stirring condition and heated to 60 °C by magnetic equipment. The product was vacuum-evaporated at 35 °C to remove excess methanol, then washed several times with distilled water and anhydrousized with Na2SO4. Samples were analyzed by GC/FID to determine the chemical composition and kept intact for later Omega-3,6,9 separation studies. 2.3. The Deep Eutectic solvents we synthezied and used for the dissertation The deep eutectic solvents synthesized for enriching and separating Omega-3,6,9 from the fat extracted from by-products in Vietnam basa fish are introduced in the table below. Table 2.3. The ratio of DES weight DES GENERATION Ration of weight (g/g) Methanol/Urea (Sample 1, Sample 2, Sample 3, Sample 4) 1:(0,14; 0,2 ; 0,23; 0,25) Choline chloride/urea (Ch/U) 1:1 Choline chloride/methylurea (Ch/MU) 1:1 Choline chloride/thiourea (Ch/Thi) 1:1 8 2.4. Synthesis of DES on the basis of choline chloride / urea and congeners The method of synthesizing DES liquid based on choline chloride was done as follows: Choline chloride and urea were put into a heat-resistant glass beaker placed on a heated magnetic stirrer in the ratio 1:1, 2:1. and 1:2 by mass and was heated at 60-70 °C with stirring until a homogeneous liquid was obtained. Experiments showed that only samples with choline chloride / urea mass ratio 1:1 and 2:1, remained liquid after cooling. The 1:2 (more urea) sample was recrystallized. Therefore, we only used samples with 1:1 ratio in follow-up studies for urea isomers of methylurea, thiourea, methylthiourea (general ratio 1:1). 2.5. Synthesis of 2-alkylbenzimidazole and DES (ethylene glycol/ benzimidazole) 2.5.1. Synthesis of 2-alkylbenzimidazole and DES (ethylene glycol/ benzimidazole) The reaction was performed between o-phenylenediamine and carboxylic acid in a ratio of 1:2. The amount of catalyst applied Choline chloride/methylthiourea (Ch/MThi) 1:1 Ethylene glycol/ 2-pentylbenzimidazole (EG/Benz-C5) 10:1,5 Ethylene glycol/ 2-heptylbenzimidazole (EG/Benz-C7) 10:1,5 Ethylene glycol/ 2-octylbenzimidazole (EG/Benz-C8) 10:1,5 Ethylene glycol/ 2-nonylbenzimidazole (EG/Benz-C9) 10:1,5 9 was 10% by weight of the substances involved in the reaction. A stream of argon gas was to expel the air from the reaction vessel. The reaction was done at a pressure of 6-8 atm, temperature 180 oC with magnetic stirrer. 2.5.2. Combination of ethylene glycol with alkylbenzimidazole to form the DES system The ethylenglycol / alkylbenzimidazole solvent system at the rate of 10-25 grams / 100 ml of ethylenglycol has been preliminarily tested. Results showed that there was not much difference in their capacity of omega separation and enrichment. However, the capacity to recover alkylbenzimidazole at the rate of 15grams / 100ml ethylenglycol was the best. The loss of alkylbenzimidazole was less than 10%. 2.6. Methods of analyzing the chemical composition of raw materials and products 2.6.1. Analysis of omega compounds by GC-FID method 2.6.2. The methods of structural analysis of DES: FTIR, GC / MS, NMR, TGA, DSC 2.6.3. Methods of Determination of the Mechanical Properties of DES. 2.6.4. Methods of Performance calculation 2.7. Method of extraction and extraction of Omega-3,6,9 from fatty acids 2.7.1. Equipment and techniques for separating Omega-3,6,9 from acids Methyl esters of fatty acids, methanol, DES were placed in a reaction vessel with continuous stirring and were heated at 45 °C. When the mixture became homogenous, they were allowed cool and then were cooled at 4 °C for 8 hours. The resulting mixture forms two 10 layers: the upper layer was a liquid, the lower layer was a solid. The solid was then washed by cold methanol. This wash solution was mixed with the original liquid layer, and let it evaporate the methanol in a vacuum evaporator and then anhydrousized. 2.7.2. The proportion of substances involved in the reactions for omega separation CHAPTER 3 FINDINGS AND DISCUSSIONS 3.1. Findings on the acid extraction from different parts of the basa fish 3.1.1. Findings on the extraction of primary fat 3.1.2. Findings on the extraction of fillets 3.1.3. Findings on the fat extraction from the by-products of Vietnamese basa fish 1029 grams of by-products obtained after filling the Vietnamese basa fish according to the process of the export seafood factores were added with water for cooking and cooling, then 86,11 grams of floating fat (accounting for 8,37% from total by-products) were obtained. Similar to catfish, 1334 grams of by-products, 163 grams of floating fat were obtained, accounting for 12,22%. Table 3.3. Findings on the fat extraction from the by-products No Obtained elements catfish (gam) basa fish (gam) 1 Fatty acid 153,69 75,34 2 Final disposals 9,24 8,023 3 loss 0,07 2,74 Total 163,00 86,11 The meat portion (fillet) used for export was only 35-37%. The fatty acids in the meat of the two kinds of fish were rounghly 11 19,8% and were equally compared. The by-products that cannot be exported accounted for a large propotion of 59-61%. After treatment, the fat recovered from catfish’ by-products were 12,22%, while that of basa fish fish were 8,37%. The amount of fatty acid obtained from the fat of catfish were 94,29% while that of Vietnamese basa fish were 87,49%. 3.2. Findings on the analysis and identification of the compounds extracted from Vietnamese basa fish 3.2.1 Findings on the analysis and identification of the primary fit 3.2.2 Findings on the analysis and identification of fat from the fillets 3.2.3 Findings on the analysis and identification of the fat from the by-products Table 3.6. Components of fat extracted from the by-products No Fat extracted from by product catfish (gam) basa fish (gam) 1 Omega-3,6,9 77,98 38,56 2 Other fatty acid 59,28 34,19 3 Triglycerides 5,06 1,78 4 Unidentified components 11,38 0,81 5 Total 153,69 75,34 The content of Omega-3,6,9 fatty acids in the fat from the by-products of catfish were 47,84%, while that of basa fish were 44,78%. Percentage of other parts of both types of fish were in the high limit from 27-34%. The ratio of Omega-3 and Omega-6 to total omega of the primary fat of basa fish reached 92,26%, proving the nutritional value. 3.2.4 Conclusions on the raw materials The content of Omega-3,6,9 compounds in catfish and basa fish parts were quite high, especially Omega-3 and Omega-6. 12 Furthermore, the by-products of these two fish contain many omega fatty acid compounds. 3.2.5. Methyl ester of the raw materials (fat from the by-products) The jelly of fatty acids was esterified to convert to methyl ester (methanol / fatty acid = 3/1, at the temperature 65 oC, in 3 hours under strong stirring conditions). Table 3.10. The contents of the compounds before and after esterification No Fatty acid Extracted jelly (%) Methyl ester (%) 1 Saturated fatty acid 31,37 35,58 2 Unsaturated fatty acid 2,85 3,35 3 Omega-3,6,9 59,15 56,97 4 Unidentified contents 6,63 4,12 Total 100 100 The chemical composition of the samples after separation and the esterification reaction with methanol were not significantly different. The total contents of Omega-3,6,9 in raw materials were about 57%; 39% were non-Omega-3,6,9 fatty acids; 4% were unknown substances. The chemical composition of the material methyl ester in the following table 3.11 would be used for the study of separation and enrichment of Omega-3,6,9 when using synthetic ionic liquids. Table 3.11. The chemical components of fatty acids in methyl ester Classification Name of compounds Methyl ester (%) Saturated fatty acids Myristic acid (14:0) 1,96 Palmitic acid(16:0) 26,55 Stearic acid (18:0) 6,78 Arachidic acid(20:0) 0,29 13 Unsaturated fatty acids Palmitoleic acid (16:1) 3,35 Omega-3 α-Linolenic acid (ALA) 18:3 (n-3) 0,46 Eicosatrienoic acid 20:3 (n-3) 0,15 Eicosapentaenoic acid (EPA) 20:5 (n-3) 0,42 Docosahexaenoic acid (DHA) 22:6 (n-3) và Nervonic acid 24:1 (n-9) 0,63 Omega-6 Linoleic acid (LA) 18:2 (n-6) 12,41 γ-Linolenic acid (GLA) 18:3 (n-6) 1,05 Eicosadienoic acid 20:2 (n-6) 0,55 Eicosatrienoic acid 20:3 (n-6) 0,18 Arachidonic acid (AA) 20:4 (n-6) 0,48 Omega-9 Oleic acid 18:1 (n-9) 40,21 Eicosenoic acid 20:1 (n-9) 0,42 Total of fatty acids 95,89 Unidentified 4,11 Total 100 3.3. The findings of DES synthesis based on choline chloride with urea and congeners 3.3.1. The findings on FTIR and TGA analysis 3.3.1.1. Choline chloride with urea FTIR νmax (KBr) cm-1: of urea 3352, 3442 cm-1 (NH), 1667 cm-1 (C = O) of amide, 1457 cm-1 (CN), of choline chloride 3376 cm-1 (OH), 3019, 2956 and 2907 cm-1 (-CH2, -CH3), 1087, 1347, 1478 cm-1 of (CO), 1643, 1206 cm-1 (CN), of choline chloride/urea all have gravels to absorb urea and choline chloride, respectively. However, the NH2 double tip of the urea has changed to a single tip because the NH2 group hydrogen bonds to hydrogen bonding with the Cl- anion, so this signal changed. In addition, the wave number of the OH group 14 in choline chloride at the 3376 cm-1 wave number was shifted to the lower region at 3347cm-1. The thermal stability of the choline chloride mixture with urea has also been checked by the TGA thermal analysis scheme to be below 200 °C. 3.3.1.2. Choline chloride with methylurea FTIR νmax (KBr) cm-1: of choline chloride 3376 cm-1 (OH), 3019, 2956 and 2907 cm-1 (-CH2, -CH3), 1087, 1347, 1478 cm-1 of (CO), 1643, 1206 cm-1 (CN), of methylurea 3344 cm-1 (NH), 2915 cm-1 (H-Csp3), 1655 cm-1 (C = O) of amide, 1353, 1171 cm-1 (CN), Choline chloride and methylurea both had absorption patterns of methylurea and choline chloride, respectively. However, the wave count of the -OH group in choline chloride at the 3376 cm-1 wave number was shifted to a lower region at 3362 cm-1. The thermal stability of the choline chloride mixture with methylurea has also been checked by thermal analysis to be below 200 °C. 3.3.1.3. Choline chloride with thiourea FTIR νmax (KBr) cm-1: of choline chloride 3376 cm-1 (O-H), 3019, 2956 and 2907 cm-1 (-CH2, -CH3), 1087, 1347, 1478 cm-1 of (CO), 1643, 1206 cm-1 (CN), of thiourea, 3376 and 1618 cm-1 (NH), 1207 cm-1 (C = S thiocarbonyl), 1413, 1084 cm-1 (CN). 2686 cm-1 (S- H), of choline chloride and thiourea, both had absorption patterns of thiourea and choline chloride, respectively. However, the wave count of the O-H group in choline chloride at the 3376 cm-1 wave number was shifted to a lower region at 3361 cm-1 and the signal strength at 2694 of the S-H junction was drastically reduced. The TGA thermal analysis diagram shows the thermal stability of the mixture of choline chloride with thiourea below 214 oC. 3.3.1.4. Choline chloride with methyl thiourea 15 FTIR νmax (KBr) cm-1: of choline chloride 3376 cm-1 (OH), 3019, 2956 and 2907 cm-1 (-CH2, -CH3), 1087, 1347, 1478 cm-1 of (CO), 1643, 1206 cm-1 (CN), of methyl thiourea 3325 and 1636 cm-1 (NH), 1302 cm-1 (C=S thiocarbonyl), 2863 cm-1 (H-Csp3), 1489, 1059 cm-1 (CN), of Choline chloride and methyl thiourea both had absorption patterns of methyl thiourea and choline chloride, respectively. However, the wave count of the O-H group in choline chloride at the 3376 cm-1 wave number was shifted to a lower region at 3324 cm-1. TGA thermal analysis diagram shows the thermal stability of the mixture below 214 oC. 3.3.2. Physical characteristics of DES based on choline chloride 3.4. Findings on the synthesis of 2-alkylbenzimidazole and the system of ethylene glycol/ benzimidazole 3.4.1. Findings on the analysis of 2-alkylbenzimidazole compounds 3.4.1.1. 2-Pentylbenzimidazole The methods of GC/MS showed the following findings: 188, 174, 160, 159, 146, 145, 133, 132 (100%), 131, 118, 92. 77, 63, 41. So với phổ dữ liệu NIST với 10 pic lớn: 132, 145, 188, 146, 159, 133, 131, 77, 63, 41. FTIR νmax (KBr) cm-1: 3082 (N-H), 2953 (C-H), 2774, 2734, 1539 (C=N), 1420, 1272(C-N), 1021, 751. 1H-NMR (CDCl3, 500 MHz, , ppm): 12,25 (1H, brs, N-H); 7,60 (2H, dd, J1=6,0 Hz, J2 = 3 Hz, H-4,7); 7,24 (2H, dd, J1 = 6,0 Hz, J2 =3,2 H-5, 6); 3,03 (2H, t, J1 = 8 Hz, J2= 7,5 Hz, H-1’); 1,91 (2H, m, H-2’); 1,36 (2H, dt, J1 =6,5 Hz, J2 = 3 Hz, H-3’); 1,27 (2H, dt, J1 = 7Hz, J2 =7 Hz, H-4’); 0,82 (3H, t, J1 = 7 Hz, J2 = 7,5 Hz, H-5’). 13C-NMR (CDCl3, 125 MHz, , ppm): , 31,4 (C-1’), 29,1 (C-2’), 28,0 16 (C-3’), 22,3 (C-4’), 13,8 (C-5’); 138,1 (C-3a,7a), 122,2 (C-5,6), 114,5 (C-4,7); 155,7 (C-2). 3.4.1.2. 2-heptylbenzimidazole The methods of GC/MS showed the following results: 216, 201, 187, 173, 160, 159, 146, 145, 133, 132, 131, 118, 92. 77, 63, 41. Compare with the NIST data spectrum of 10 large pics: 132, 145, 131, 187, 146, 216, 159, 133, 77, 63, 41. FTIR νmax (KBr) cm-1: 3086 (N-H), 2954, 2927 (C-H), 2740, 1541 (C=N), 1449, 1423, 1273 (C-N), 1028, 751. 1H-NMR (CDCl3, 500 MHz, , ppm): 12,86 (1H, brs, N-H); 7,63 (2H, dd, J1 = 3 Hz, J2 = 3 Hz, H-4,7); 7,27 (2H, dd, J1 = 3 Hz, J2 = 3 Hz, H- 5,6); 3,08 (2H, t, J1 = 7,5 Hz, J2 = 8 Hz, H-1’); 1,95 (2H, m, H-2’); 1,4 (2H, m, H-3’); 1,23 (6H, m, H-4’,5’,6’); 0,85 (3H, t, J1 = 7Hz, J2 = 7 Hz, H-7’). 13C-NMR (CDCl3, 125 MHz, , ppm): , 31,6 (C-1’), 29,3 (C-2’), 29,2 (C-3’), 28,9( C-4’), 28,5 (C-5’), 22,5 (C-6’), 13,9 (C-7’); 138,4 (C-3a,7a), 122,0 (C-5,6), 114,5 (C-4,7); 156 (C-2). 3.4.1.3. 2-Octylbenzimidazole The methods of GC/MS showed the following results: 230, 215, 201, 187, 173, 146, 145, 132, 131, 118, 92, 77, 63, 41. Compare with the NIST data spectrum of 10 large pics: 230, 215, 201, 187, 159, 146, 145 , 132 ,131, 83, 41. FTIR νmax (KBr) cm-1: 2927, 2856 (C-H), 2677, 1538 (C=N), 1436, 1419, 1273 (C-N), 1002, 840, 752. 1H-NMR (CDCl3, 500 MHz, , ppm): 11,87 (1H, brs, N-H); 7,58 (2H, dd, J1 = 3 Hz, J2 = 3 Hz, H-4,7); 7,23 (2H, dd, J1 = 3 Hz, J2 = 3 Hz, H-5,6); 3,02 (2H, t, J1 = 8 Hz, J2 = 7,5 Hz, H-1’); 1,9 (2H, m, H-2’); 1,36 (2H, m, H-3’); 1,2 (8H, m, H-4’,5’,6’,7’); 0,84 (3H, t, J1 = 7 Hz, J2 = 7 Hz, H-8’). 13C-NMR (CDCl3, 125 MHz, , ppm): 31,7 17 (C-1’), 29,3 (C-2’), 29,2 (C-3’), 29,1( C-4’), 28,4 (C-5’,6’), 22,5 (C- 7’), 13,9 (C-8’); 138,2 (C-3a), 138,1 (C-7a), 122,0 (C-5,6), 114,5 (C- 4,7); 156 (C-2). 3.4.1.4. 2-Nonylbenzimidazole The methods of GC/MS showed the following results: 244, 229, 215, 201, 187, 173, 160, 159, 146, 145, 133, 132 (100%), 131, 118, 92. 77, 63, 41. Compare with the NIST data spectrum of 10 large pics:132, 145, 244, 187, 146, 131, 201,118, 77,41. FTIR νmax (KBr) cm-1: 3088 (N-H), 2926, 2853 (C-H), 2771, 1542 (C=N), 1454, 1422, 1272 (C-N), 1028, 752. 1H-NMR (CDCl3, 500 MHz, , ppm): 12,32 (1H, brs, N-H); 7,6 (2H, dd, J1 = 3,5 Hz, J2 = 3 Hz, H-4,7); 7,24 (2H, dd, J1 = 3 Hz, J2 = 3 Hz, H-5,6); 3,04 (2H, t, J1 = 8 Hz, J2 = 7,5 Hz, H-1’); 1,92 (2H, m, H-2’); 1,37 (2H, m, H-3’); 1,24 (10H, m, H-4’,5’,6’,7’,8’); 0,87 (3H, t, J1 = 7 Hz, J2 = 7,5 Hz, H-9’). 13C-NMR (CDCl3, 125 MHz, , ppm): 31,8 (C-1’), 29,4 (C-2’,3’), 29,3 (C-4’), 29,2( C-5’), 28,4 (C- 6’,7’), 22,5 (C-8’), 14,1 (C-9’); 138,1 (C-3a,7a), 122,3 (C-5,6), 114.5 (C-4,7); 156 (C-2). 3.4.2. Ethylene glycol / alkylbenzimidazole solution system 2-Alkylbenzimidazole are solids with melting points of 2- pentylbenzimidazole, 2-heptylbenzimidazole, 2-octylbenzimidazole, 2-nonylbenzimidazole: 167 oC, 150 oC, 143 oC and 133 oC respecti- vely. Therefore, to use them in omega extraction, we had to use a solution of ethylene glycol /benzimidazole at the rate of 10 /1,5 (1,5 grams of 2-alkylbenzimidazole dissolved in 10 grams of EG). 3.5. Result of enriching and separating Omega-3,6,9 from the methyl ester fatty acid mixture of raw materials with deep eutectic solvent (DES) 18 3.5.1. Separation and enrichment of Omega-3,6,9 by the methanol / urea system 3.5.1.1. The results of the methanol / urea enrichment system after mixing with methyl ester 3.5.1.2. Results of phase separation and liquid chemical composition of methanol / urea The results showed that the urea concentration in DES solution at 0,2 g / ml gave the best liquid composition efficiency of 36%. As the urea concentration increased, the liquid fraction decreased. Meanwhile, the urea concentration in the solution was lower than 0,2g / ml, the effect of forming the liquid component is not high (23% when the urea concentration is 0,143 g / ml). Table 3.18. The weight of the liquids after extraction Classification ME Sample 1 Sample 2 Sample 3 Sample 4 % grams % grams % grams % grams % grams FA 35,58 7,12 12,58 0,58 6,08 0,44 8,13 0,47 9,48 0,47 UFA 3,35 0,67 2,55 0,12 2,67 0,19 3,12 0,18 2,47 0,13 Omega-3 1,66 0,33 3,32 0,15 4,26 0,31 3,91 0,23 3,86 0,19 Omega-6 14,67 2,93 45,88 2,11 31,25 2,25 32,76 1,90 31,69 1,58 Omega-9 40,63 8,13 31,35 1,44 53,20 3,83 49,08 2,85 49,50 2,48 Unidentified 4,11 0,82 4,32 0,20 2,54 0,18 3,0 0,17 3,0 0,15 Total 100 20 100 4,6 100 7,2 100 5,8 100 5,0 Solvent system with urea concentration of 0,2g / ml was the best for separation performance and content of substances such as EPA increased 2 times, DHA increased 3 times. In conclusion: 1. When urea was allowed to mix directly with methyl ester of fatty acids, the phenomenon of urea changed the structure of these 19 fatty acids. When using urea in the form of a deep eutectic solvent with a urea concentration of ½ saturation point could separate Omega-3,6,9 from fatty acids with a purity up to 88,71%, with an efficiency of 36% for one split times. 2. Substances such as ETA and EPA were both 1,5 to 3 times higher than the starting materials. Especially in sample 2, DHA content increased 3 times. Total Omega-3 accounts for 5% of the Omega-3,6,9 mixture separated. 3. When using methanol and urea deep eutectic solvents with a concentration of 0,2 g /ml higher or lower, the separation efficiency of compounds decreased and the omega content also decreased. 3.5.2. Separation and enrichment of Omega-3,6,9 by the choline chloride system 3.5.2.1. The results of enrichment of choline chloride/urea and homologous systems 3.5.2.2. Results of phase separation and liquid chemical composition of choline chloride / urea and homologous systems Table 3.25. The weight of the liquidy products after separation of choline chloride system Classification ME Ch/U Ch/ MU Ch/Thi Ch/MThi grams % grams % grams % grams % grams % FA 7,12 35,58 0,11 3,29 1,49 41,32 1,07 37,57 0,80 29,00 UFA 0,67 3,35 0,08 2,36 0,11 3,19 0,05 1,82 0,09 3,58 Omega-3 0,37 1,66 0,15 4,34 0,19 5,37 0,22 7,47 0,57 20,54 Omega-6 2,89 14,67 1,10 33,40 0,55 15,26 0,49 17,40 1,01 36,67 Omega-9 8,13 40,63 1,76 53,29 1,12 30,99 0,90 31,79 0,26 9,23 Unidentified 0,82 4,11 0,10 3,32 0,14 3,87 0,11 3,95 0,03 0,98 Total ME 20 100 3,30 100 3,60 100 2,84 100 2,76 100 Omega-3,6,9 11,39 56,96 3,01 91,03 1,86 51,62 1,61 56,66 1,84 66,44 20 Separation efficiency of choline chloride/urea and choline chloride/methyl urea systems reached 17 and 18%, respectively. The remaining two solvents were in the 14% range. Although the total Omega-3,6,9 of the choline chloride and urea solvents after blending decreased by ½ compared to the starting material, 50% of the Omega-3,6,9 in the mixture were separated out with the concentration up to 91%. Other solvents, despite an Omega- 3,6,9 content being 2 times higher than that of the choline chloride and urea systems, were only able to separate about 15% and the Omega-3,6,9 content in the fraction only. reach 51-66%. Summary 1. The choline chloride/urea system (and its congeners) did not change the structure of compounds in methyl ester, but the ability to separate Omega-3,6,9 is high, especially the choline chloride/urea system with phase split was 16,5% and the Omega-3,6,9 content was up to 91%. 2. The efficiency of separating saturated fatty acids and unsaturated fatty acids was over 96%. 3. The total content of ALA, EPA, DHA went up to 20% in separated liquid products. 3.5.3. Separation and enrichment of Omega-3,6,9 with the ethylene glycol / benzimidazole system 3.5.3.1. The results of enrichment of ethylene glycol / benzimidazole systems 3.5.3.2. Results of phase separation and liquid chemical composition of ethylene glycol / benzimidazole systems - The omega-enrichment and separation performance of alkylbenzimidazole was only 15% of the material in one time. 21 - The saturated fatty acids in the liquid portion was significantly reduced, especially when using 2-pentylbenzimidazole, 2-heptylbenzimidazole and 2-octylbenzimidazole but the content was still high (from 13-24% depending on the alkyl circuit). Saturated fatty acids and unsaturated fatty acids were still quite high, nearly 10%, only the EG / Benz-C9 system remained 20%. - The content of Omega-3,6,9 in the extracted solution was not high, only reaching 87% for EG / Benz-C5, Benz-C7, Benz-C8 and 76% for EG / Benz-C9. Table 3.32. The weight of liquid product after extraction of EG/Benz Classification ME Benz-C5 Benz-C7 Benz-C8 Benz-C9 grams % grams % grams % grams % grams % FA 7,12 35,58 0,23 7,56 0,18 5,74 0,19 6,18 0,51 16,86 UFA 0,67 3,35 0,11 3,65 0,11 3,41 0,11 3,59 0,15 4,86 Omega-3 0,37 1,66 0,82 27,66 0,73 23,64 0,99 31,93 1,43 47,16 Omega-6 2,89 14,67 0,08 2,82 0,04 1,26 0,04 1,33 0,54 17,63 Omega-9 8,13 40,63 1,69 56,59 1,93 62,31 1,66 53,68 0,34 11,10 Unidentified 0,82 4,11 0,05 1,72 0,11 3,64 0,11 3,29 0,07 2,39 Total ME 20 100 2,98 100 3,10 100 3,10 100 3,04 100 Omega-3,6,9 11,39 56,96 2,59 87,07 2,7 87,21 2,69 86,94 2,31 75,89 Summary: This deep eitectic solvent system had a similar high efficiency in separating saturated and unsaturated fatty acids, but the separation efficiency of omega fatty acids was low. For Omega-3, the highest EG/Benz-C8 system could reach the efficiency of 20%, while the other systems were only about 15-17%. However, this system led to a significant increase in the amount of Omega-3s, mainly α- Linolenic acid (ALA), up to 41% (8 grams per 20 grams ME) after mixing and stirring. Unfortunately, the capacity to separate Omega-3 22 from the solution was not high. It is necessary to have further studies to coordinate systems that are both able to produce more Omega-3, and be able to separate them with higher efficiency. 3.6. Comparison and evaluation of Omega-3,6,9 enrichment efficiency of deep eutectic solvent systems 3.6.1. Capacity to enrich Omega-3,6,9 when it was not separated Omega-3,6,9 enrichment was performed in two main stages: The first stage was to mix and stir ingredients at low temperature until the mixture was homogeneous. The next stage was to cool at room temperature then cooled to 4 °C for 8 hours, after this phase the mixture split into 2 layers. Figure 3.40. Some potentialities for a Linolenic acid α-formation Methanol/urea system (sample 2): After mixing, the content of saturated fatty acids and unsaturated fatty acids decreased 23 dramatically 6,77 grams. Meanwhile, Omega-3,6.9 increased a total of 6,89 grams T