misnistry of education and training vietnam academiy of science and technology

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 requireme[r]

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: 44 01 14

A SUMMARY OF THE DISSERTATION ON ORGANIC CHEMISTRY

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:

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

special properties such as non-volatility and no vaporized pressure Therefore, they 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

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-octylben2-heptylben-zimidazole, 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

CHAPTER 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

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

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

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 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

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 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

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

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

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

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

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

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 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

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)

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 (-CH

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 (-CH

2, -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 (-CH

2, -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

FTIR νmax (KBr) cm-1: of choline chloride 3376 cm-1 (OH), 3019, 2956 and 2907 cm-1 (-CH

2, -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 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 (CDCl

3, 500 MHz, , ppm): 12,25 (1H, brs, N-H); 7,60 (2H, dd, J1=6,0 Hz, J2 = Hz, H-4,7); 7,24 (2H, dd, J1 = 6,0 Hz, J2 =3,2 H-5, 6); 3,03 (2H, t, J1 = Hz, J2= 7,5 Hz, H-1’); 1,91 (2H, m, H-2’); 1,36 (2H, dt, J1 =6,5 Hz, J2 = Hz, H-3’); 1,27 (2H, dt, J1 = 7Hz, J2 =7 Hz, H-4’); 0,82 (3H, t, J1 = Hz, J2 = 7,5 Hz, H-5’) 13C-NMR (CDCl

(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 (CDCl

3, 500 MHz, , ppm): 12,86 (1H, brs, N-H); 7,63 (2H, dd, J1 = Hz, J2 = Hz, H-4,7); 7,27 (2H, dd, J1 = Hz, J2 = Hz, H- 5,6); 3,08 (2H, t, J1 = 7,5 Hz, J2 = 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 = 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

1’), 29,3 2’), 29,2 3’), 29,1( C-4’), 28,4 5’,6’), 22,5 7’), 13,9 8’); 138,2 3a), 138,1 7a), 122,0 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 = Hz, H-4,7); 7,24 (2H, dd, J1 = Hz, J2 = Hz, H-5,6); 3,04 (2H, t, J1 = 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 = Hz, J2 = 7,5 Hz, H-9’) 13C-NMR (CDCl3, 125 MHz, , ppm): 31,8 1’), 29,4 2’,3’), 29,3 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

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)

3.5.1 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 Sample Sample Sample

% 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 times, DHA increased times

In conclusion:

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 times higher than the starting materials Especially in sample 2, DHA content increased 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

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 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 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

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 °C for hours, after this phase the mixture split into layers

dramatically 6,77 grams Meanwhile, Omega-3,6.9 increased a total of 6,89 grams

The choline chloride/urea system (other isomers) did not significantly affect the chemical composition of the raw methyl ester Some changes increased or decreased the volume of substances, but not by much

The ethylene glycol/benzimidazole system had significantly enriched Omega-3 The conversion to Omega-3 mainly went from Omega-6 and Omega-9 Saturated fatty acids and unsaturated fatty acids in the EG/Benz-C5, EG/Benz-C7, EG/Benz-C8 systems converted only 26% to 28% and 30%, respectively, EG/Benz-C9 hardly converted saturated fatty acids and unsaturated fatty acids 3.6.2 The capacity of phase separation (liquid and solid)

Except for the methanol/urea system (sample 2) which had the best phase separation performance the other systems were in the range of 15-18% for a single split Although the choline chloride/urea system was only about 16,5%, the total Omega-3,6,9 in this system was the highest, at 91%

3.6.3 The capacity to separate compounds in the liquid contents Except for the methanol/urea system, other DES systems could separate saturated fatty acids from 80-96%, the Ch/U system alone could reach 99% The separation efficiency of Omega-3,6,9 compounds of DES systems was based on the mass produced by mixing with the mass separated from the liquid contents

Table 3.38 Omega-3,6,9 extraction performance in the liquidy content

Omega-3 Omega-6 Omega-9

DES Forma tion (g) Separa tion (g) perfor mance (%) Forma tion (g) Separa tion (g) perfor mance (%) Forma tion (g) Separa tion (g) perfor mance (%) M-1 0,64 0,15 23,44 9,2 2,11 22,94 6,28 1,44 22,93 M-2 0,86 0,31 36,05 6,68 2,25 33,68 10,74 3,83 35,66 M-3 0,78 0,23 29,48 6,95 1,90 27,34 10,54 2,85 27,04 M-4 0,88 0,19 21,59 7,63 1,58 20,70 10,14 2,48 24,46 Ch/U 0,23 0,15 65,21 1,87 1,10 58,82 4,01 1,76 43,89 Ch/MU 0,64 0,19 29,69 3,01 0,55 18,27 8,12 1,12 13,79 Ch/Thi 0,63 0,22 34,92 2,89 0,49 16,96 8,23 0,90 10,94

Ch/MThi 0,91 0,57 62,64 3,51 1,01 28,77 6,94 0,26 3,75

EG/BC-5 4,76 0,82 17,22 1,52 0,08 5,26 7,00 1,69 24,14

EG/BC-7 5,05 0,73 14,45 1,49 0,04 2,68 7,23 1,93 26,69

EG/BC-8 5,07 0,99 19,52 1,40 0,04 2,86 6,72 1,66 24,70

EG/BC-9 8,22 1,43 17,40 1,80 0,54 30,00 1,68 0,34 20,24

outer layer, from solid to semi-solid Over time under low temperature conditions, the part coalesced and separated from non-"solvates" components This mechanism helps the choline chloride and urea-containing systems created saturated fatty acids, unsaturated fatty acids as well as other fatty acids into the solid part

3.6.4 Comparison of Omega-3 quality separated in liquid products Omega-3 in products extracted from DES systems were mainly ETA, ALA, EPA, DHA With regular content at ≤ 2% Particularly, in the case of using the ethylene glycol / benzimidazole system, the ALA content increased significantly from 20-44%

Figure 3.46 Omega-3 structure f the liquidy contents in DES 3.6.5 General conclusion about DES systems

The methanol/urea system was a simple, inexpensive system, but the content of Omega-3,6,9 after separation was only 80 to 88%

CONCLUSIONS 1 Research on raw materials

- Traditional solvents were used to extract fatty acids from parts of basa fish and catfish The portion of meat used for export were only 35-37% The by-products account for quite a large percent of 59-61% After treatment, the fat derived from catfish by-products was 12,22%, while that of basa fish was 8,37% The amount of fatty acid in the derived fat of catfish was 94,29% while that of basa fish was 87,49%

- Omega compounds in primary fats, meat and fats from basa fish and catfish by-products were identified The amount of Omega-3,6,9 in the by-products of basa fish and catfish was 5,13% and 6,12% The raw materials used for the extraction and enrichment of Omega-3,6,9 in the by-products of the methylester include saturated fatty acid: 35,58%; unsaturated fatty acids: 3,35%; Omega-3: 1,66%; Omega-6: 14,67%; Omega-9: 40,63%

2 Successful synthesis of deep eutectic solvents (DES)

- The solvent system methanol/urea with concentrations (g/ml) 0,143; 0,2; 0,23; 0,26 were successfully synthesized

- The solvent system based on choline chloride including choline chloride/urea and congeners (methylurea; thiourea and methylthiourea) were successfully synthesized

3 Using the deep eutectic solvent systems synthesized to separate and enrich Omega-3,6,9 from basa fish fish waste fat

- Three synthetic deep eutectic solvent systems were used to enrich and separate Omega-3,6,9 from the methylester in raw materials Separation efficiency was from 15-36% for one time of extraction with Omega-3,6,9 content reaching from 52-91% depending on the DES system

4 Comparison of the omega enrichment and split capacity of the DES systems

- The methanol/urea system (sample 2) had the best phase separation efficiency of 36% The other systems were in the range of 15-18% for a single split However, for choline chloride urea system was only about 16,5%, but Omega-3,6,9 content in this system was high, from 57% of raw materials to 91%, which is a new finding Other DES systems could separate saturated fatty acids at 80-96%, especially choline chloride/urea system reached 99%

CONTRIBUTIONS BY THE DISSERTATION a / In terms of scientific values

- Three deep eutectic solvents have been successfully synthesized and applied for the first time to enrich and separate Omega-3,6,9 from by-products derived from processing Vietnamese basa fish procedures for export in Dong Thap

- A new finding is that the use of the ethylene glycol/benzimidazole deep eutectic solvent system showed that this system made the content of α-Linolenic acid (ALA) 18: (n-3) (a form of Omega-3 quite important) increase by 44%

- The use of DES type of deep eutectic solvents to separate and enrich Omega-3,6,9 from the waste fat of Vietnamese basa fish so as to obtain the new Omega-3,6,9 whose content reached more than 90% and the Omega-3,6 content in the separated products reached from 22-47%

b / In term of economic values

LIST OF THE AUTHOR’S PUBLISHED ARTICLES

1 Thanh Xuan Le Thi, Hoai Lam Tran, Thanh Son Cu, and Son Lam Ho, Separation and Enrichment of Omega 3, 6, and Fatty Acids from the By-Products of Vietnamese basa fish Fish Processing using Deep Eutectic Solvent, Journal of Chemistry Hindawi, 2018, doi.org/10.1155/2018/6276832, IF=1.75

2 The patent for useful solution, No 2419, The procedures for extracting and enriching Omega 3,6,9 from fat abotained from the by-products in processing Vietnamese basa fish for export by using Deep Eutectic Solvents, Department of Copyright and Intellectuals of Vietnam, Decision No 11375w/QĐ-SHTT, dated 12/08/2020

3 Lê Thị Thanh Xuan, Nguyen Minh Thao, Cu Thanh Son, Ho Son Lam, Survey composition and content of Omega-3,6,9, extracted from catfish at Mekong Delta in Vietnam by extraction method with the traditional solvents, Journal of Chemistry Viet Nam, 2017, 5e34, 55, 551-556

4 Le Thi Thanh Xuan, Le Thi Hoa Xuan, Cu Thanh Son, Ho Son Lam, Indentifying the composition of Omega-3,6,9 content in Vietnamese basa fish in Mekong delta, Journal of Chemistry and Application Viet Nam, 2018, 4, 59-63