A summary of the dissertation on Organic chemistry: Synthesis of some deep eutectic solvents based on 2-alkylbenzimidazole, choline chloride and their application to extract Omega. 3,6,9 in Vietnamese basa fish in Mekong Delta

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The goals of the dissertation: Synthesizing deep eutectic solvents based on choline chloride with the compounds of 2-alkylbenzimidazole/ethylene glycol and determining their structures; using synthetic deep eutectic solvents to enrich and separate Omega-3,6,9 from waste fat in accordance with the facrories’ procedure of processing Vietnamese basa fish for export.

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Nội dung Text: A summary of the dissertation on Organic chemistry: Synthesis of some deep eutectic solvents based on 2-alkylbenzimidazole, choline chloride and their application to extract Omega. 3,6,9 in Vietnamese basa fish in Mekong Delta

MISNISTRY OF VIETNAM ACADEMIY OF EDUCATION AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY LE THI THANH XUAN 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 Major: Organic chemistry Code: 9 44 01 14 A SUMMARY OF THE DISSERTATION ON ORGANIC CHEMISTRY HO CHI MINH CITY – 2021
This dissertation is completed in: Graduate University of Science and Technology – Vietnam Academy of Science and Technology Supervisor 1: Ho Son Lam, Asso, Prof. Dr Supervisor 2: Cu Thanh Son, Dr This dissertation will be defended by the Committee of Dissertation Evaluation at institutional level, at Graduate University of Science and Technology – Vietnam Academy of Science and Technology at…....., 2021 This dissertation could be accessed at: - The library of Graduate University of Science and Technology - National Library of Vietnam
1 INTRODUCTION Fish raising industry, especially that of Vietnamese basa fish, is under strong development in the Mekong Delta, meeting domestic consumption demand and serving as raw processing materials for export, promoting the development of Vietnam's seafood industry. However, we have used Vietnamese basa fish mainly their meat (fillet) for export and primary fats for the domestic market. By- products of the processing process such as head, bones, fat, skin... have not been utilized effectively but used as a fertilizer or animal feed instead. If there is no appropriate treatment, we will not only waste the nutrients contained in it, but also leave them as sources of pollution to the environment. In recent years, in Vietnam, there have been a large number of studies on the separation of omega compounds by such methods as: Hydrolysis of lipids in alkaline environment and urea precipitation or using the thermal fractionation method at high level… to improve the economic efficiency of the catfish processing. However, the projects are only exploratory, without any research work on separating Omega-3,6,9 compounds from the fatty acid mixture of Vietnamese basa fish and fish fat. In the world, the Omega-3,6,9 extraction methods available in research and production include chromatographic method, enzyme and distillation method, urea complex precipitation method, super liquid extraction method. Deep eutectic solvents are a newly-generated ionic liquid that can be generated by mixing the appropriate ingredients and then heating them to produce liquid with a lower freezing point than the initial individual ingredients. The freezing point lowering is the result of hydrogen bonding agent interactions. Deep eutectic solvents have
2 special properties such as non-volatility and no vaporized pressure. Therefore, they do not pose problems related to fire or explosion, are safe in transport or to the habitat, as well as biodegradability and biocompatibility. They can replace toxic solvents, limit environmental pollution, and are able to be recovered and reused. They are, therefore, the prioritized elements and used as solvents in extraction techniques. Separation of Omega-3,6,9 from the fatty acid mixture as well as the separation of each type of omega with a green and low- cost solvent system is a common matter concerned by researchers and manufacturers. Based on the actual demand for Omega-3,6,9 in Vietnam, as well as the need to improve the economic efficiency of Vietnamese basa fish, meeting the high scientific requirements of separation and enrichment Omega-3,6,9 by using green and inexpensive solvents, we have proposed the project "Synthesis of some deep eutectic solvents based on 2-alkylbenzimidazole, choline chloride and their application to extract Omega. 3,6,9 in Vietnamese basa fish in Mekong Delta” and it was accepted by the Graduate Academy of Science and Technology as my graduate dissertation. * The goals of the dissertation - Synthesizing deep eutectic solvents based on choline chloride with the compounds of 2-alkylbenzimidazole / ethylene glycol and determining their structures. - Using synthetic deep eutectic solvents to enrich and separate Omega-3,6,9 from waste fat in accordance with the facrories’ procedure of processing Vietnamese basa fish for export. * The contents of the dissertation
3 1. Investigating the composition and fatty acid content of Vietnamese basa fish by traditional solvents and building the fatty acid separation process from the by-products gained from the Vietnamese basa fish fillet processing stages. 2. Experimenting and synthesizing Deep Eutectic Solvent by using: - Choline chloride with urea and isoforms (methylurea, thiourea and methylthiourea) to determine their properties. - 2-Alkylbenzimidazole (2-pentylbenzimidazole, 2-heptylben- zimidazole, 2-octylbenzimidazole, 2-nonylbenzimidazole), and creating a deep eutectice solvent system ethylene glycol / 2-alkylbenzimidazole. 3. Using the synthetic deep eutectic solvents to separate and enrich Omega-3,6,9 from raw materials. 4. Comparing the enrichment and separation of Omega-3,6,9 by the synthesized deep eutectic solvent. * The scientific and practical significance of the dissertation Finding out a method of separating and enriching Omega-3,6,9 from by-products of Vietnamnese basa fish with deep eutectic solvents for high omega content. Making effective use of by-products derived from processing Vietnamese basa fish for export in Mekong Delta. Contributing to enhancing the economic value of fish and protecting the environment. This research orientation is quite new and of high academic value because in Vietnam there has been no research on separating and enriching Omega-3,6,9 from fat from the by-products derived from the processing of Vietnamese basa fish for export by deep eutectic solvents.
4 CHAPTER 1 INTRODUCTION 1.1 Brief introduction to ionic liquid 1.2 Deep eutectice solvent – a new generated liquid (DES) 1.2.1. Brief introduction to DES – formation and development In the early of 20th century, a new ionic liquid system was generated by mixing quaternary ammonium salt (2-hydroxyethyl trimethyl ammonium chloride) with several hydrogen-bonding agents such as amide, glycol or carboxylic acid (mono- diacid). They form a solvent capable of dissolving several salts and metal oxides, which is also of low cost and easy to recycle or decompose without polluting the environment. This new ionic liquid is called DES (deep eutectic solvent). The first publications on this ionic liquid system were done by Abbott and his co-researchers since 2001. To highlight DES's growing interest in green solvents, C. Andrew and other researchers sought through the cited database of Web of Science Citation Database for many research works in the field of DES. When the compounds making up DES are the main ones, such as amino acids, organic acids, sugars or choline derivatives, DES is known as deep natural eutectic solvents (NADES). In terms of physical and chemical properties, NADES fully feature the green chemical principles. 1.2.2. Scientific background of DES According to Abbott [64], when a solid organic salt and a complex agent are mixed in the right ratio and heated, they will produce a liquid with a lower freezing point than the initial individual ingredients. In addition, the temperature of the eutectic mixture was below the boiling point of water. Lower freezing point is a result of
5 hydrogen bonding interaction between the complexing agent and the organic salt. Hydrogen bond is a very weak bond formed by electrostatic attraction between hydrogen (bonded in a molecule) and a strong electronegative atom of small size (N, O, F ...) in another molecule or in the same molecule. Hydrogen bonds can be formed between molecules or within the same molecule. 1.3. Introduction to Vietnamese basa fish and the content of Omega-3,6,9 in the fish 1.3.1. Introduction to Vietnamese bas fish 1.3.2. Potentialities of Vietnamese basa fish and content of omega in the fish 1.4. Economic values and application of Omega-3,6,9 in our lives 1.4.1. Research works on the pharmaceautical values of omega to human beings 1.4.2. Introduction to fat 1.4.3. Classification of fat 1.4.4. Introduction to omega 1.5. Several extraction methods for Omega-3,6,9 available in research and production They include chromatographic method, enzyme and distillation method, low-temperature crystallization or precipitation of complex urea, supercritical liquid. Vietnam has a number of research projects to improve the economic efficiency of the processing of Vietnamese basa fish as well as by-products from the fish, such as hydrolysis and precipitation by urea for unsaturated fatty acids, using the fractional crystallization method at low temperature for the Omega-3 content of 22,8-24,05% [109,110], the lipid hydrolysis method in alkaline and precipitation medium urea
6 precipitate to obtain fatty acids including saturated fatty acids and unsaturated fatty acids with omega content of 24,5% [111]. There are also authors [112] using traditional solvents to extract omega from Vietnamese basa fish fat and performing transesterification to analyze and determine their composition. In general, these claims are only exploratory, there is no research on separating Omega-3,6,9 compounds from the fatty acid mixture of Vietnamese basa fish by deep eutectic solvents. CHAPTER 2 EXPERIMENTAL PROCIDURES 2.1. Chemical elements, raw materials and experimental tools 2.2. Preparation of Vietnamese basa fish 2.2.1. The 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 Weight Percentage Weight Percentage No Components (gam) (%) (gam) (%) 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, 1:(0,14; 0,2 ; 0,23; 0,25) Sample 4) Choline chloride/urea (Ch/U) 1:1 Choline chloride/methylurea (Ch/MU) 1:1 Choline chloride/thiourea (Ch/Thi) 1:1
8 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 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
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 catfish basa fish No Obtained elements (gam) (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 catfish basa fish No Fat extracted from by product (gam) (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 Extracted jelly Methyl ester No Fatty acid (%) (%) 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 (%) Myristic acid (14:0) 1,96 Saturated fatty Palmitic acid(16:0) 26,55 acids Stearic acid (18:0) 6,78 Arachidic acid(20:0) 0,29
13 Unsaturated Palmitoleic acid (16:1) 3,35 fatty acids α-Linolenic acid (ALA) 18:3 (n-3) 0,46 Eicosatrienoic acid 20:3 (n-3) 0,15 Omega-3 Eicosapentaenoic acid (EPA) 20:5 (n-3) 0,42 Docosahexaenoic acid (DHA) 22:6 (n-3) và 0,63 Nervonic acid 24:1 (n-9) Linoleic acid (LA) 18:2 (n-6) 12,41 γ-Linolenic acid (GLA) 18:3 (n-6) 1,05 Omega-6 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 Oleic acid 18:1 (n-9) 40,21 Omega-9 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. 1 H-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’). 13 C-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. H-NMR (CDCl3, 500 MHz, , ppm): 12,86 (1H, brs, N-H); 1 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’). 13 C-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. 1 H-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’). 13 C-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’). 13 C-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 ME Sample 1 Sample 2 Sample 3 Sample 4 Classification % 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