tatiana sofia marçalo vaz bifásicos extraction of ... de antioxidantes com... · problemas devido...
TRANSCRIPT
Universidade de Aveiro
2014
Departamento de Química
Tatiana Sofia Marçalo Vaz
Extração de antioxidantes com sistemas aquosos bifásicos
Extraction of antioxidants with aqueous biphasic systems
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Universidade de Aveiro
2014 Departamento de Química
Tatiana Sofia Marçalo Vaz
Extração de antioxidantes com sistemas aquosos bifásicos
Extraction of antioxidants with aqueous biphasic systems
Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Bioquímica Clínica, realizada sob a orientação científica da Doutora Mara Guadalupe Freire Martins, Investigadora Coordenadora no Departamento de Química, CICECO, da Universidade de Aveiro, e da Doutora Isabel Maria Boal Palheiros, Professora Auxiliar no Departamento de Química da Universidade de Aveiro.
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Aos meus pais e ao meu maninho...
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O júri
presidente Professor Doutor Pedro Miguel Dimas Neves Domingues
Professor Auxiliar no Departamento de Química da Universidade de Aveiro
Doutora Mara Guadalupe Freire Martins
Investigadora Coordenadora no Departamento de Química, CICECO, da Universidade de Aveiro
Doutora Carmen Sofia da Rocha Freire Barros
Investigadora Principal no Departamento de Química, CICECO, da Universidade de Aveiro
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agradecimentos
Antes de mais gostaria de agradecer às minhas orientadoras, Mara Freire e Isabel Boal por todo o apoio e excelente acompanhamento ao longo deste trabalho. Agradeço a todos os membros do grupo path e mini-path. Um especial obrigado à Mafalda, Ana Maria e Matheus, pela paciência que tiveram comigo. Estiveram sempre disponíveis para tirar todas as minhas dúvidas, ajudaram-me sempre que precisei e deram-me muito apoio nos dias em que parecia que tudo corria mal. Claro, sem esquecer a Maria João; trabalhos muito, desesperamos muito mas principalmente divertimo-nos muito durante este percurso. Também à Andreia quero agradecer por estar sempre disponível para me apoiar e me animar nos dias menos bons. Aos meus tios quero agradecer por tudo o que fizeram por mim, estão sempre disponíveis para me apoiar em tudo. Avó, obrigado por acreditares tanto em mim. Um especial obrigado aos meus pais, sem vocês nada disto seria possível, quero agradecer por todo o amor, por sempre acreditarem em mim e me apoiarem incondicionalmente em todas as minhas decisões. Vocês são os melhores. Sem esquecer os meus dois meninos mais lindos do mundo, Kevim e André. Adoro-vos. Rafael, obrigado pela paciência que tiveste comigo, acreditaste sempre em mim e deste-me todo o teu apoio.
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palavras-chave
Antioxidantes, sistemas aquosos bifásicos, extração, líquidos iónicos, polímeros
resumo
Atualmente os antioxidantes apresentam uma relevância elevada numa vasta gama de aplicações utilizadas pela indústria farmacêutica e de cosmética. Os antioxidantes são capazes de inibir ou de retardar a oxidação de outros compostos, principalmente a oxidação causada por radicais livres, e daí o seu destaque na indústria e da sua incorporação nos produtos mais variados. No entanto, para a utilização de antioxidantes em quantidades eficazes, é necessário sintetizá-los quimicamente, o que é dispendioso e pode acarretar problemas devido à sua origem não natural, ou extraí-los das suas fontes naturais (biomassa). Os métodos convencionais para a extração e posterior purificação de antioxidantes são normalmente dispendiosos e morosos. Neste sentido, têm vindo a ser estudados outros métodos de extração, e mais recentemente, a extração líquido-líquido recorrendo a sistemas aquosos bifásicos (SAB). Neste trabalho propõe-se a utilização de SAB constituídos por líquidos iónicos (LIs) e polímeros como alternativa aos sistemas convencionais maioritariamente formados por polímeros e sais inorgânicos. A utilização de LIs permite afinar as propriedades dos sistemas extrativos por variação dos iões que os compõem, e portanto, aumentar as eficiências de extração. Neste trabalho foi demostrada, pela primeira vez, a formação de SAB constituídos por polivinilpirrolidona (PVP) e álcool polivinílico (PVA) de várias massas moleculares e LIs das famílias dos fosfónios e dos imidazólios. Os respetivos diagramas de fase foram determinados a 25 ºC de modo a inferir sobre as concentrações mínimas necessárias para a formação de duas fases aquosas. Por fim, estes sistemas foram avaliados quanto à sua capacidade como sistemas de extração do tipo líquido-líquido. Para tal, determinaram-se os coeficientes de partição e as eficiências de extração para vários antioxidantes (ácido gálico, vanilina, ácido vanílico e ácido siríngico) utilizando os diversos sistemas estudados. Obtiveram-se eficiências de extração que variaram entre 75 % e 95 %, o que demonstra a viabilidade destes sistemas na extração de antioxidantes. De um modo geral, o sistema mais eficiente para extrair antioxidantes é composto pelo LI 1-butil-3-metilimidazólio trifluorometanosulfonato ([C4mim][CF3SO3]) juntamente com o PVA de 9.000-10.000 g.mol-1 (PVA9) para todos antioxidantes estudados.
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keywords
Antioxidants, aqueous biphasic systems, extraction, ionic liquids, polymers
abstract
Nowadays, antioxidants present a great relevance in a variety of applications used by the pharmaceutical and the cosmetic industries. The antioxidants are able to inhibit or to retard the oxidation of other compounds, and mainly the oxidation caused by free radicals, and that is the reason of their importance in the industry and their incorporation into a variety of products. For the utilization of antioxidants in effective quantities, it is necessary to either chemically synthesize them, what is expensive and may causes problems due to their non-natural origin, or to extract them from their natural sources (biomass). Conventional methods for the extraction of antioxidants and the following purification are normally expensive and time-consuming. Therefore, other extraction methods, like the liquid-liquid extraction using aqueous biphasic systems (ABS), have been studied. In this work, the use of ABS based on ionic liquids (ILs) and polymers is proposed as an alternative for the conventional systems that are mostly composed of polymers and inorganic salts. The use of ILs allows the tuning of the properties of the extraction systems by variation of their ions and so to increase the extraction efficiencies. In this work, it is shown, for the first time, that poly(vinyl pyrrolidone) (PVP) and poly(vinyl alcohol) (PVA) of different molecular weights form ABS with phosphonium-based and imidazolium-based ILs. The respective phase diagrams were determined at 25 °C to define the minimum concentrations that are necessary for the formation of two aqueous phases. Finally, the ability of these systems as liquid-liquid extraction systems was evaluated. To this end, the partition coefficients and extraction efficiencies of several antioxidants (gallic acid, vanillin, vanillic acid and syringic acid) using the studied systems were determined. Extraction efficiencies ranging from 75% to 95 % were obtained for the different systems, thus demonstrating their capability for extracting purposes. In general, the most efficient system for the extraction of antioxidants was that composed of the IL 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([C4mim][CF3SO3]) and the PVA of 9,000-10,000 g.mol-1 (PVA9) for the extraction of all the studied antioxidants. .
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LIST OF CONTENTS
1.General introduction ........................................................................................................ 1
1.1. Objectives and scope ............................................................................................. 3
1.2. Antioxidants .......................................................................................................... 5
1.2.1. Gallic acid, syringic acid, vanillic acid and vanillin ..................................... 6
1.2.2. Mechanisms of action .................................................................................... 8
1.2.3. Importance of antioxidants in physiological aging process and disease ..... 10
1.2.4. Cosmetic applications.................................................................................. 13
1.4. Extraction of antioxidants ................................................................................... 15
1.4.1. Solid-liquid extraction ................................................................................. 16
1.4.1.1 Supercritical fluid extraction method (SFE) ......................................... 16
1.4.1.2. Accelerated solvent extraction (ASE) ................................................. 17
1.4.1.3. Ultra-sound assisted extraction (UAE) ................................................ 17
1.4.1.4. Microwave assisted extraction (MAE) ................................................ 18
1.4.2. Comparison of solid-liquid extraction methods for gallic, syringic, and
vanillic acids and vanillin ...................................................................................... 18
1.5. Liquid-liquid extraction ...................................................................................... 20
1.5.1. Aqueous Biphasic Systems (ABS) .............................................................. 22
1.5.2. Ionic liquids (ILs) ........................................................................................ 25
1.5.3. Polymers ...................................................................................................... 30
2.IL + PVP + H2O ABS ..................................................................................................... 35
2.1. Poly(vinyl pyrrolidone) (PVP) ............................................................................ 37
2.2 Experimental section ............................................................................................ 40
2.2.1. Chemicals .................................................................................................... 40
2.2.2. Experimental procedure .............................................................................. 44
2.2.2.1. Phase diagrams and tie-lines ................................................................ 44
2.2.2.2. pH measurements................................................................................. 47
2.2.2.3. Partition of antioxidants ....................................................................... 47
2.3. Results and discussion ........................................................................................ 48
2.3.1. Phase diagrams and tie-lines ....................................................................... 48
2.3.2. Partition of antioxidants .............................................................................. 61
2.4. Conclusions ......................................................................................................... 69
3.IL + PVA + H2O ABS ..................................................................................................... 71
3.1. Poly(vinyl alcohol) (PVA) .................................................................................. 73
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3.2. Experimental section ........................................................................................... 75
3.2.1. Chemicals .................................................................................................... 75
3.2.2. Experimental procedure .............................................................................. 75
3.2.2.1 Phase diagrams and tie-lines ................................................................. 75
3.2.2.2 Partition of antioxidants ........................................................................ 76
3.3. Results and discussion ........................................................................................ 77
3.3.1. Phase diagrams and tie-lines ....................................................................... 77
3.3.2. Partition of antioxidants .............................................................................. 86
3.4. Conclusions ......................................................................................................... 94
4.Final remarks .................................................................................................................. 95
4.1. Conclusions ......................................................................................................... 97
4.2. Future work ......................................................................................................... 97
5.References........................................................................................................................ 99
6.Publications ................................................................................................................... 111
Appendix A Calibration curves .............................................................................. 115
Appendix B Experimental binodal data ................................................................. 119
Appendix C Experimental data for partitioning of antioxidants ........................... 127
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LIST OF TABLES
Table 1.1- Properties of gallic acid, vanillic acid, vanillin and syringic acid [15]. .............. 7
Table 2.1- Cation an anion structure and molecular weights of the ILs studied. ............... 42
Table 2.2- Combinations of the ILs and PVPs of different molecular weights that are able
(√) or not able, because of completely miscibility (m) or precipitation (p) to form ABS. .. 49
Table 2.3- Correlation parameters used to describe the experimental binodal data by
Equation 1 for the ABS based on PVP and ILs. .................................................................. 57
Table 2.4- Experimental data for TLs of the ABS IL + PVP + H2O. ................................. 60
Table 2.5- Average pH values of the bottom (pHPVP) and the top (pHIL) phase of the ABS
used for the extraction of the antioxidants........................................................................... 61
Table 3.1- Combinations of the ILs and PVAs of different molecular weights that are able
(√) or not able, because of completely miscibility (m) or precipitation (p), to form ABS. . 78
Table 3.2- Correlation parameters used to describe the experimental binodal data by
Equation 1 for the ABS based on IL and PVA. ................................................................... 84
Table 3.3- Experimental data for TLs of the ABS IL + PVA + H2O. ................................ 86
Table 3.4- Average pH values of the PVA-rich (pHPVA) and the IL-rich (pHIL) phase of the
ABS used for the extraction of the antioxidants .................................................................. 87
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LIST OF FIGURES
Figure 1.1- Classification of antioxidants in different groups. Adapted from [13]. ............. 6
Figure 1.2- Chemical structures of gallic acid, vanillic acid, vanillin and syringic acid...... 7
Figure 1.3- Reaction chain of the free radicals. Adapted from [12]. .................................... 9
Figure 1.4- Involvement of antioxidants in the free radicals reaction chain. Adapted from
[12]. ..................................................................................................................................... 10
Figure 1.5- Phase diagram of an ABS composed of PEG and a salt aqueous solution [106].
............................................................................................................................................. 23
Figure 1.6- Ternary phase diagram of an ABS composed by an ionic liquid and an inorganic
salt aqueous solution [105]. ................................................................................................. 24
Figure 1.7- Chemical structure of common IL cations and anions..................................... 26
Figure 1.8- Number of published articles regarding ILs per year (in the last 20 years).
Information taken from ISI Web of Knowledge in 17th January 2014. ............................... 27
Figure 1.9-Molecular structures of (i) PEG/PEO, (ii) PPG and (iii) PVA. ........................ 31
Figure 2.1- Chemical structure of PVP. ............................................................................. 37
Figure 2.2- Experimental determination of the phase diagrams. (A) Cloudy solution
(biphasic region); (B) Clear solution (monophasic region). ................................................ 45
Figure 2.3- Antioxidant extraction with an ABS composed of IL + PVP + H2O. ............. 47
Figure 2.4- Phase diagrams for the ternary systems composed of IL + PVP10 + H2O at 25
°C in mol.kg-1 units (left) and in wt.% (right): ▲, [P4444]Br; ♦, [P4441][CH3SO4]; ■,
[P4442][Et2PO4]; ▲, [Pi(444)1][Tos]. ...................................................................................... 50
Figure 2.5- Phase diagrams for the ternary systems composed of IL + PVP29 + H2O at 25
°C in mol.kg-1 units (left) and in wt.% (right): ▲, [P4444]Br; ♦, [P4441][CH3SO4]; ■,
[P4442][Et2PO4]; ▲, [Pi(444)1][Tos]. ...................................................................................... 51
Figure 2.6- Phase diagrams for the ternary systems composed of IL + PVP40 + H2O at 25
°C in mol.kg-1 units (left) and in wt.% (right): ▲, [P4444]Br; ●, [P4444]Cl; ♦, [P4441][CH3SO4];
■, [P4442][Et2PO4]; ▲, [Pi(444)1][Tos]. .................................................................................. 52
Figure 2.7- Phase diagrams for ternary systems composed of [P4444]Br + PVP + H2O at 25
°C in mol.kg-1 units (left) and in wt.% (right): ♦, PVP10; ■, PVP29; ▲, PVP40. ............ 54
Figure 2.8- Phase diagrams for ternary systems composed of [P4441][CH3SO4] + PVP + H2O
at 25 °C in mol.kg-1 units (left) and in wt.% (right): ♦, PVP10; ■, PVP29; ▲, PVP40. .... 54
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Figure 2.9- Phase diagrams for ternary systems composed of [P4442][Et2PO4] + PVP + H2O
at 25 °C in mol.kg-1 units (left) and in wt.% (right): ♦, PVP10; ■, PVP29; ▲, PVP40. .... 55
Figure 2.10- Phase diagrams for ternary systems composed of [Pi(444)1][Tos] + PVP + H2O
at 25 °C in mol.kg-1 units (left) and in wt.% (right): ♦, PVP10; ■, PVP29; ▲, PVP40. .... 55
Figure 2.11- Phase diagram for the ternary system composed of [P4444]Br + PVP40 + H2O:
binodal curve data (▲); TL 1 data (X); TL 2 data (●); adjusted binodal data through
Equation 1 (―). ................................................................................................................... 58
Figure 2.12- Phase diagram for the ternary system composed of [P4442][Et2PO4] + PVP40 +
H2O: binodal curve data (▲); TL 1 data (X); TL 2 data (●); adjusted binodal data through
Equation 1 (―). ................................................................................................................... 58
Figure 2.13- Partition coefficients of gallic acid, vanillin, vanillic acid and syringic acid in
a biphasic system composed of 67 wt.% IL + 13 wt.% PVP40 + 20 wt.% antioxidant aqueous
solution at 25 °C and average pH values of the IL-rich phase (Top) and PVP-rich phase
(Bottom). ............................................................................................................................. 62
Figure 2.14- Extraction efficiency of gallic acid, vanillin, vanillic acid and syringic acid in
a biphasic system composed of 67 wt. % of IL + 13 wt.% of PVP40 + 20 wt. % of antioxidant
aqueous solution at 25 °C and pH values of the IL-rich phase (Top) and PVP-rich phase
(Bottom). ............................................................................................................................. 63
Figure 2.15- Partition coefficient of gallic acid, vanillin, vanillic acid and syringic acid and
the pH values of the IL-rich phase (pH Top) and the PVP-rich phase (pH Bottom) in a
biphasic system composed of 67 wt.% [P4444]Br + 13 wt.% PVP + 20 wt.% antioxidant
aqueous solution at 25 °C. ................................................................................................... 66
Figure 2.16- Extraction efficiency of gallic acid, vanillin, vanillic acid and syringic acid and
the pH values of the IL-rich phase (pH Top) and the PVP-rich phase (pH Bottom) in a
biphasic system composed of 67 wt.% [P4444]Br + 13 wt.% PVP + 20 wt.% antioxidant
aqueous solution at 25 °C. ................................................................................................... 67
Figure 3.1- Chemical structure of PVA. ............................................................................. 73
Figure 3.2- Antioxidant extraction with an ABS composed of IL + PVA + H2O. ............. 76
Figure 3.3- Precipitation of the system composed of IL + PVA + H2O. ............................ 79
Figure 3.4- Phase diagrams for the ternary systems composed of [C4mim][CF3SO3] + PVA
+ H2O at 25 °C in mol.kg-1 units (left) and in wt.% (right): ■, PVA85; ▲, PVA30; x, PVA13;
♦, PVA9. .............................................................................................................................. 80
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Figure 3.5- Phase diagrams for the ternary systems composed of [C4mim][BF4] + PVA +
H2O at 25 °C in mol.kg-1 units (left) and in wt.% (right): ■, PVA85; ▲, PVA30; x, PVA13;
♦, PVA9. .............................................................................................................................. 81
Figure 3.6- Phase diagrams for the ternary systems composed of IL + PVA9 + H2O at 25
°C in mol.kg-1 units (left) and in wt.% (right): ■, [C4mim][BF4]; ♦, [C4mim][CF3SO3]. ... 82
Figure 3.8- Phase diagrams for the ternary systems composed of IL + PVA30 + H2O at 25
°C in mol.kg-1 units (left) and in wt.% (right): ■, [C4mim][BF4]; ♦, [C4mim][CF3SO3]. ... 83
Figure 3.9- Phase diagrams for the ternary systems composed of IL + PVA85 + H2O at 25
°C in mol.kg-1 units (left) and in wt.% (right): ■, [C4mim][BF4]; ♦, [C4mim][CF3SO3]. ... 83
Figure 3.10- Phase diagram for the ternary system composed of [C4mim][BF4] + PVA30 +
H2O: binodal curve data (▲); TL 1 data (X); TL 2 data (●); adjusted binodal data through
Equation 1 (―). ................................................................................................................... 85
Figure 3.11- Phase diagram for the ternary system composed of [C4mim][CF3SO3] + PVA13
+ H2O: binodal curve data (▲); TL 1 data (X); TL 2 data (●); adjusted binodal data through
Equation 1 (―). ................................................................................................................... 85
Figure 3.12- Partition coefficients of gallic acid in a biphasic system composed of IL +PVA
+ antioxidant solution at 25 °C and average pH values of the IL-rich phase (Bottom) and
PVA-rich phase (Top). ........................................................................................................ 88
Figure 3.13- Extraction efficiency of gallic acid in a biphasic system composed of IL +PVA
+ antioxidant solution at 25 °C and average pH values of the IL-rich phase (Bottom) and
PVA-rich phase (Top). ........................................................................................................ 89
Figure 3.14- Partition coefficients of gallic acid, syringic acid, vanillin and vanillic acid in
a biphasic system composed of 58 wt. % of [C4mim][CF3SO3] + 3 wt. % of PVA9 + 39 wt.
% of antioxidant solution at 25 °C and average pH values of the IL-rich phase (Bottom) and
PVA-rich phase (Top). ........................................................................................................ 91
Figure 3.15- Extraction efficiency of gallic acid, syringic acid, vanillin and vanillic acid in
a biphasic system composed of 58 wt. % of [C4mim][CF3SO3] + 3 wt. % of PVA9 + 39 wt.
% of antioxidant solution at 25 °C and average pH values of the IL-rich phase (Bottom) and
PVA-rich phase (Top). ........................................................................................................ 92
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LIST OF SYMBOLS
KOW – octanol-water partition coefficient (dimensionless);
R2 – correlation coefficient (dimensionless);
α – ratio between the top weight and the total weight of the mixture (dimensionless);
[IL] – concentration of ionic liquid (wt % or mol·kg-1);
[IL]IL – concentration of ionic liquid in ionic-liquid-rich phase (wt %);
[IL]PVP – concentration of ionic liquid in poly(vinyl pirrolidone)-rich phase (wt %);
[IL]M – concentration of ionic liquid in mixture point (wt %);
[PVP] – concentration of poly(vinyl pirrolidone) (wt % or mol·kg-1);
[PVP]IL – concentration of poly(vinyl pirrolidone) in the ionic-liquid-rich phase (wt
%);
[PVP]PVP – concentration of poly(vinyl pirrolidone) in poly(vinyl pirrolidone)-rich
phase (wt %);
[PVP]M – concentration of poly(vinyl pirrolidone) at the mixture point (wt %);
[AO]IL – concentration of antioxidant in the ionic-liquid-rich phase (wt %);
[AO]PVP – concentration of an antioxidant in the poly(vinyl pirrolidone)-rich
phase (wt %);
KAO – partition coefficient of an antioxidant (dimensionless);
EEAO% – percentage extraction efficiencies of an antioxidant (%).
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ACRONYMS
ABS - Aqueous biphasic system;
AD - Alzheimer disease;
ASE - Accelerated solvent extraction;
DNA - Deoxyribonucleic acid;
FDA – U.S. Food and Drug Administration
IL - Ionic liquid;
LLE - Liquid-liquid extraction;
LMWA - Low molecular weight antioxidant;
MAE - Microwave assisted extraction;
MMP - Matrix metalloproteinases;
NFT - Neurofibrillary tangles;
PEG - Poly(ethylene glycol);
PEO – Poly(ethylene oxide);
PPG - Poly(propylene glycol);
PVA - Poly(vinyl alcohol);
PVA9 - Poly(vinyl alcohol) of 9,000-10,000 g.mol-1;
PVA13 - Poly(vinyl alcohol); of 13,000-23,000 g.mol-1;
PVA30 - Poly(vinyl alcohol) of 30,000-70,000 g.mol-1;
PVA85 - Poly(vinyl alcohol) of 85,000-124,000 g.mol-1;
PVAc - Poly(vinyl acetate);
PVP - Poly(vinyl pyrrolidone);
PVP10 - Poly(vinyl pyrrolidone) of 10,000 g.mol-1;
PVP29 - Poly(vinyl pyrrolidone) of 29,000 g.mol-1;
PVP40 - Poly(vinyl pyrrolidone) of 40,000 g.mol-1;
RNA - Ribonucleic acid;
RNS - Reactive nitrogen species;
ROS - Reactive oxygen species;
RTIL - Room temperature ionic liquid;
SFE - Supercritical fluid extraction;
TL - Tie-line;
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TLL - Tie-line length;
UAE - Ultra-sound assisted extraction;
UV - Ultraviolet;
VOC - Volatile organic compound;
[amim]Cl – 1-allyl-3-methylimidazolium chloride;
[C2mim]Br – 1-ethyl-3-methylimidazolium bromide;
[C2mim]Cl – 1-ethyl-3-methylimidazolium chloride;
[C2mim][CF3SO3] - 1-ethyl-3-methylimidazolium trifluoromethanesulfonate;
[C2mim][CH3CO2] - 1-ethyl-3-methylimidazolium acetate;
[C2mim][HSO4] - 1-ethyl-3-methylimidazolium hydrogensulfate;
[C2mim][PO4(CH3)2] - 1-ethyl-3-methylimidazolium dimethylphosphate;
[C4mim][BF4] - 1-butyl-3-methylimidazolium tetrafluoroborate;
[C4mim]Br - 1-butyl-3-methylimidazolium bromide;
[C4mim]Cl – 1-butyl-3-methylimidazolium chloride;
[C4mim][C2H5SO4] - 1-butyl-3-methylimidazolium ethylsulfate;
[C4mim][CF3SO3] - 1-butyl-3-methylimidazolium trifluoromethanesulfonate;
[C4mim][CH3CO2] - 1-butyl-3-methylimidazolium acetate;
[C4mim][CH3SO3] - 1-butyl-3-methylimidazolium methanesulfonate;
[C4mim][CH3SO4] - 1-butyl-3-methylimidazolium methylsulfate;
[C4mim][N(CN)2] - 1-butyl-3-methylimidazolium dicyanamide;
[C4mim][OctSO4] - 1-butyl-3-methylimidazolium octylsulfate;
[C4mim][PO4(CH3)2] - 1-butyl-3-methylimidazolium dimethylphosphate;
[C4mim][SCN] - 1-butyl-3-methylimidazolium thiocyanate;
[C4mim][Tos] - 1-butyl-3-methylimidazolium tosylate;
[C4mpip]Cl - 1-butyl-3-methypiperidinium chloride;
[C4mpyr]Cl - 1-butyl-3-methypyrrolidinium chloride;
[C6mim]Cl – 1-hexyl-3-methylimidazolium chloride;
[C6mim][N(CN)2] - 1-hexyl-3-methylimidazolium dicyanamide;
[C7H7mim]Cl – 1- benzyl-3-methylimidazolium chloride;
[C7mim]Cl – 1-heptyl-3-methylimidazolium chloride;
[C8mim]Cl – 1-octyl-3-methylimidazolium chloride;
[C10mim]Cl – 1-decyl-3-methylimidazolium chloride;
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[CH3CH2NH3][NO3] – Ethylammonium nitrate;
[Ch][Bic] – cholinium bicarbonate;
[Ch][Bit] – cholinium bitartrate;
[Ch][CH3CO2] – cholinium acetate;
[Ch]Cl – cholinium chloride;
[Ch][DHCit] – cholinium dihydrogencitrate;
[Ch][DHPh] – cholinium dihydrogenphosphate;
[Ch][Lac] – cholinium lactate;
[Ch][Prop] – cholinium propionate;
[N1120][CH3CO2] – N,N-dimethyl-N-ethylammonium acetate;
[N11[2(N110)]0][CH3CO2] – N,N-dimethyl-N-(N´,N´-dimethylaminoethyl) ammonium
acetate;
[N11[2(N110)]0]Cl – N,N-dimethyl-N-(N´,N´-dimethylaminoethyl) ammonium chloride;
[OHC2mim]Cl – 1-hydroxyethyl-3-methylimidazolium chloride;
[P4441][CH3SO4] – tributyl(methyl)phosphonium methylsulfate;
[P4442][Et2PO4] – ethyl(tributyl)phosphonium diethylphosphate;
[P4444]Br – tetrabutylphosphonium bromide;
[P4444]Cl – tetrabutylphosphonium chloride;
[Pi(444)1][Tos] – triisobutyl(methyl)phosphonium tosylate.
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1
1. General introduction
2
3
1.1. Objectives and scope
Antioxidants have been a target of intense research since it is acknowledged that they
can retard or inhibit the damages that oxidative stress can cause to cells and their
components, like lipids, proteins, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
and that may even lead to cell death [1]. Antioxidants can thus be described as a defense
system against species that cause oxidative stress.
The damages caused by oxidative stress are also responsible for some diseases, such
as Alzheimer’s disease (AD), Parkinson’s disease, atherosclerosis, cancer and arthritis [1].
In this context, antioxidants can play a crucial role on pharmacological applications in the
prophylaxis or therapy of these diseases. Antioxidants have also cosmetic usage, either for
preventing the oxidation of some products in cosmetics formulations or as ingredients of
anti-aging treatment products [2].
Antioxidants may either be chemically synthesized or extracted from natural sources.
The conventional methods for the extraction of antioxidants, like maceration, percolation
and soxhlet extraction have some disadvantages, since most of them are time consuming
require the use of organic, volatile and toxic solvents (and thus malignant for the human
health and the environment) and are also expensive [3,4]. The soxhlet extraction is the
mostly applied method and has been used for many years. Nevertheless, it cannot be used for
the extraction of compounds that are sensitive to high temperatures because this technique
is carried out at the boiling point of the solvents and for a long time and, therefore, the
compounds can suffer decomposition [4]. Hence, new and alternative methods have been
put forward to replace the conventional ones, such as the supercritical fluid extraction (SFE),
accelerated solvent extraction (ASE), ultra-sound assisted extraction (UAE) and microwave
assisted extraction (MAE). All these methods are solid-liquid-type extractions, and thus, the
purification step that follows consists generally in a liquid–liquid extraction (LLE) aiming
at achieving a high effectiveness, high yield, improved purity degree, proper selectivity,
technological simplicity, and low cost [5]. These LLE processes conventionally use
immiscible solvents; however, due to the detrimental character of most organic solvents,
improvements have been made in this approach. Among those, attempts have been carried
out employing aqueous biphasic systems (ABS) to avoid the use of organic and volatile
solvents in the extraction/purification stage [6].
4
In recent years, the use of ABS consisting of two immiscible aqueous phases has
emerged as a more biocompatible, environmentally-friendly and economically feasible
technique for the extraction/purification of several compounds [6]. ABS are formed when
two water soluble compounds form two mutually immiscible aqueous phases; above a
critical concentration of those components, a spontaneous phase separation takes place. The
extraction of distinct biomolecules can be achieved by the manipulation of their affinity for
each phase. But the quest to find new ways, more cost-effective, sustainable and
environmentally friendly systems still goes on aiming at improving the development of ABS.
The main objective of this work is to study the ability of ABS for the extraction and/or
purification of antioxidants. For this purpose, ABS based on ionic liquids (ILs) and
polymers, that have advantageous properties comparatively to the commonly used systems
majorly composed of ILs and inorganic salts, are here investigated. Several ILs were
investigated, namely phosphonium-, cholinium-, ammonium- and imidazolium-based
combined with the most diverse anions. Phosphonium-based compounds were the most
deeply investigated since they are chemically and thermally more stable, more benign and
biocompatible than other ILs [7,8]. The polymers studied were poly(vinyl pyrrolidone)
(PVP) and poly(vinyl alcohol) (PVA), both benign and biofriendly polymers, as new phase-
forming components of ABS involving ILs.
Initially, the phase diagrams, tie-lines and tie-line lengths of the diverse ABS were
determined at 25 ºC. These phase diagrams provide crucial indications on the minimum
phase-forming components amounts required to create an ABS. Additionally, these systems
were evaluated in what concerns their extraction ability (by means of partition coefficients
and extraction efficiencies) for different antioxidants, namely gallic acid, vanillic acid,
vanillin and syringic acid.
5
1.2. Antioxidants
Antioxidants are compounds that are able to retard or inhibit the oxidation of other
compounds. Even though oxygen is very important for surviving, it can also be harmful for
cells if it is present in high concentrations, forming reactive oxygen species (ROS) that can
be radical or non-radical agents. The radicals are molecules that bear an unpaired electron
on the outer shell [9], which makes them very reactive, and so they can initiate reactions
with other molecules generating more radicals.
In normal conditions, there is a balance between the oxidant and antioxidant species
but, when there is an increase of the oxidant or a decrease of the antioxidant species, the
unbalance can generate oxidative stress [10]. The oxidative stress can cause damages into
cells and/or its components, like proteins, lipids, DNA and RNA, that may lead to loss of
function and even to the death of cells [1]. Antioxidants are a very important combat system
against the damages caused by oxidative stress. Antioxidants can be classified in two major
groups, namely the enzymatic and non-enzymatic antioxidants, as shown in Figure 1.1.
The enzymatic antioxidants can be divided into primary and secondary enzymes. The
primary enzymes, such as glutatione peroxidase, catalase and superoxide dismutase
constitute the primary defense system against free radicals; they inhibit the formation of the
radicals or neutralize them. The secondary enzymes, like glutathione reductase and glucose-
6-phosphate dehydrogenase, that are the secondary defense system, cannot directly
neutralize the free radicals but support the action of other antioxidants [11].
The non-enzymatic antioxidants can be dividing into 7 groups, namely carotenoids,
low molecular weight antioxidants, organosulfur compounds, minerals, cofactors, vitamins
and polyphenols; in the latter group are included the flavonoids and the phenolic acids [12].
Antioxidants may also be divided into endogenous, that are synthesized in the
organism, or exogenous, that must be ingested as food or medication. The endogenous
antioxidants may be enzymatic, like glutathione peroxidase, catalase and superoxide
dismutase or non-enzymatic, like uric acid and albumin. In some situations, the endogenous
antioxidants are not sufficient to combat the oxidative stress and then it is necessary to
complement them with exogenous antioxidants, from supplements or pharmaceuticals, but
also from food rich in antioxidants, such as vitamin A, vitamin E and ß-carotene [13].
6
Figure 1.1- Classification of antioxidants in different groups. Adapted from [12].
1.2.1. Gallic acid, syringic acid, vanillic acid and vanillin
Among the phenolic antioxidants are gallic acid, syringic acid, vanillic acid and
vanillin; their chemical structures are shown in Figure 1.2. All the four phenolic compounds
have similar structures; vanillin differs from vanillic acid because it bears an aldehyde
instead of an acid group. Vanillic acid differs from the syringic acid because it has only one
methoxyl group and the syringic acid has two of them. Finally, gallic acid has two hydroxyl
groups at the same position where the syringic acid has the methoxyl groups.
Antioxidants
enzymatic
Primary enzymes
Secondary
enzymes
Non enzymatic
Polyfenols
Flavonoids
Phenolic acids
Vitamins
Cofactors
Minerals
Organosulfurcompounds
Low molecular weight molecules
Carotenoids
Polyphenols
Enzymatic
7
Figure 1.2- Chemical structures of gallic acid, vanillic acid, vanillin and syringic acid.
In Table 1.1 are presented the chemical formula, the molecular weight, the density, the
melting point, the dissociation constant (pKa), the octanol-water partition coefficient (Kow)
and the solubility in water of the four phenolic compounds mentioned before [14].
Table 1.1- Properties of gallic acid, vanillic acid, vanillin and syringic acid [14].
Gallic acid Vanillic acid Vanillin Syringic acid
Chemical formula C7H6O5 C8H8O4 C8H8O3 C9H10O5
Molecular weight
(g/mol) 170.12 168.15 152.15 198.17
Density (g.cm-3) 1.7 1.4 1.2 1.3
Melting point (°C) 253 211.5 81.5 206-209
pKa 3.94; 9.04;
11.17 4.16; 10.14 7.81 3.93; 9.55
log (Kow) 0.70 1.43 1.21 1.04
Solubility in water at
25ºC (g/L) 58.95 3.77 11.00 5.78
Gallic acid or 3,4,5-trihydroxybenzoic acid is a white crystalline powder that is
included in the group of phenolic acids. It can be abundantly found in red wine, gallnuts,
grapes, tea and other plants [15]. Gallic acid has not only antioxidant properties [16] but it
also has anti-inflammatory [17], anti-cancer [18], anti-mutagenic [19], anti-angiogenic [20],
anti-bacterial and anti-viral [21] activities. This compound has an antioxidant activity as a
chelator and it is a scavenger of free radicals. It protects the neurons from damages and
apoptosis caused by ß-amyloid [22] and the lymphocytes from the lipid peroxidation and
DNA fragmentation [23]. Gallic acid can further inhibit the saturation of lipids [24]. It can
also protect the cardiovascular system by inhibiting the NO production and so inhibiting
Gallic acid vanillic acid vanillin syrinic acidsyringic acidgallic acid vanillic acid vanillin syringic acid
8
vasorelaxation [25]. Finally, it was already demonstrated that it has higher ability to induce
apoptosis in tumor cells than in normal cells [26,27].
Vanillic acid or 3-methoxy-4-hydroxybenzoic acid is a white compound in the form
of powder or crystals and belongs to the group of phenolic acids. It is an oxidized form of
vanillin. It is abundantly found in vanilla beans [28,29] and is able to inhibit the snake venom
activity [30,31], carcinogenesis [32], apoptosis [33,34] and inflammation [35]. It is used in
fragrances and as a food additive.
Vanillin or 3-methoxy-4-hydroxybenzaldehyde is a phenolic aldehyde. Alike vanillic
acid, it is used in fragrances and as a food additive. It is the primary extraction compound of
vanilla beans [28]. It displays antioxidant [36,37], antibacterial [38] and anticarcinogenic
activities [39].
The syringic acid or 3,5-dimethoxy-4-hydroxybenzoic acid is a phenolic acid that is
used as a sedative and local anesthetic, with antitussive and expectorant effects and it is
widely used as a medicine for bronchitis [40]. Recently, syringic acid was demonstrated to
show strong antioxidant, antiproliferative [41], anti-endotoxic [42], anti-cancer [43] and
hepatoprotective [35] activities.
1.2.2. Mechanisms of action
It is not possible to assign a sole mechanism of action for all antioxidants because there
is a large number of antioxidant species and they do not act exactly through the same way.
In addition, antioxidants do not act alone; instead, they interfere in each other’s activity and
can act synergistically. Consequently, the mechanism of action and the efficiency of
antioxidants depend on their localization and on other antioxidants that they interact with,
among other factors [44,45].
Low molecular weight antioxidants (LMWAs) are small molecules capable to pass
through the cell membranes [46]; they can enter the cells and inhibit the oxidative stress and
are regenerated by the cell afterwards [9]. They can be synthesized in the organism - the
endogenous ones, or be obtained from diet - the exogenous LMWAs.
The free radicals that cause oxidative stress can be oxygen derived such as the reactive
oxygen species (ROS), like superoxide, hydroxyl and peroxyl, or nitrogen derived, the
reactive nitrogen species (RNS), like nitric oxide, peroxy nitrate and nitrogen dioxide. The
9
excess of free radicals can generate a chain of reactions with the following steps: initiation,
propagation, branching and termination [47]. This chain of reactions is summarized in Figure
1.3, where an example for the oxidation of lipids caused by the radicals is shown. In Figure
1.4 it is depicted how the antioxidants can stop this reaction chain. The antioxidants that are
able to stop this reaction chain are for example phenolic acids, like the gallic acid, vanillic
acid, syringic acid and also the vanillin. However, external agents, like heat, light or ionizing
radiation are factors that can initiate this reaction chain [48].
Figure 1.3- Reaction chain of the free radicals. Adapted from [11].
10
Figure 1.4- Involvement of antioxidants in the free radicals reaction chain. Adapted from [11].
In Figure 1.3 it is shown that in the initiation step a substrate (LH), a lipid in this
example, can be attacked by a free radical (R∙) and forms a very reactive alkyl radical (L∙).
In the propagation phase this alkyl radical, due to its reactivity, can react with oxygen and
form a lipid peroxyl radical (LOO∙), which further reacts with other lipids to form more alkyl
radicals. This propagation can lead to the formation of much more radicals. In this stage,
lipid hydroperoxides (LOOH) are also formed, and which can be converted in the branching
phase in other compounds, such as alcohols, aldehydes, ketones and radicals, such as alkoxy
radicals (LO∙) [49]. The termination of this reaction chain only occurs when two radicals
react with each other to form a non-radical molecule.
Antioxidants (AH) can stop the reaction chain summarized in Figure 1.3 and so prevent
the formation of more radicals. The antioxidants can inhibit the initiation by reacting with
the lipid radical or the propagation by reacting with the alkoxyl or the lipid peroxyl radical
[50]. In a first step the antioxidant became a radical itself that is more stable and less reactive
than the other free radicals. In a following step the antioxidant radical reacts with another
reactive radical and donate its electron (Figure 1.4).
Secondary antioxidants do not act directly with the reaction chain but can retard this
process by removing the substrates or by quenching oxygen [47,51].
1.2.3. Importance of antioxidants in physiological aging process and disease
Aging is a very complex process and with the increase of age there are changes in the
human organism that make it more susceptible for diseases and death to occur, either due to
molecular or genetic changes [52]. There are many theories about the causes of the aging
11
process - an accepted one is that the physiological changes on aging are a consequence of
the damages caused to DNA by oxidative stress [49]. The oxygen present in the cells can
induce the initiation of reactions that can lead to damages in the biomolecules present in the
cells, such as proteins, lipids, carbohydrates and DNA. There are evidences that the decline
of the skeletal muscle with aging is also caused by oxidative damages [53,54].
Considering the evidences that aging is caused by oxidative stress and that antioxidants
can combat it, then the aging-associated conditions of human body can be retarded by using
antioxidants [55]. Harman [56] proposed, for the first time in 1956, that physiological aging
is related to the damages caused by oxidative stress to the cells and their components. The
author also concluded that neurodegenerative diseases associated with aging are caused by
oxidative stress [56]. Later on, in 1972, the author explained that the mitochondria is the
primary responsible for the physiological aging [57], because it was identified that oxidative
stress is higher in cells and their components with higher consumption of oxygen
(mitochondria is the cell component with the highest consumption of oxygen). Also, the
antioxidant system is affected by age [58,59] due to the increase on oxidative stress and
decrease of the mitochondrial function.
Oxidative stress may be also responsible for the development of many diseases due to
the damages caused into cells by ROS, such as Alzheimer’s disease, Parkinson’s disease,
atherosclerosis, cancer and arthritis [1]. Epidemiological studies have shown that the
consumption of foods, like vegetables and fruits, can give a good protection against diseases
associated with oxidative stress due to the antioxidants that they contain [60]. It is not
possible to generalize the effects of antioxidants through all the diseases, because every
disease has a different mechanism which the oxidative stress affects and the antioxidants
protect us from these mechanisms at a different way in each disease.
As an example, the role of the antioxidants in Alzheimer disease (AD) is here
considered. The Alzheimer disease is a neurodegenerative disease that has a progressive
evolution; the patients usually have loss of memory and dementia. At a molecular level, AD
is characterized by neurofibrillary tangles (NFT), which are intraneuronal filamentous
inclusions, and the extracellular senile plaques and the loss of neurons and synapses. In AD
there is the accumulation of ß-amyloid and hyperfosforilated Tau proteins in the brain that
cause these NFTs and senile plaques [61]. The accumulated ß-amyloid induces the
lipoperoxidation of membranes and lipid peroxidation products [62]. The Tau protein is a
12
protein that is present in the neurons in high quantity; with oxidative stress this protein is
hyperfosforilated and can lose its function or gain toxic function and this may lead to neuron
death [63]. Also the DNA and RNA are oxidized by oxidative stress which leads also to
neuronal death [62].
If the conditions associated with the oxidative stress are considered as a major cause
of AD, then antioxidants can be used to retard or maybe treat this disease. And in fact, many
epidemiological and clinical studies for the effects of antioxidants lead to the conclusion that
the ingestion of a high quantity of antioxidants can retard the progression of the disease [64]
and to diminish the risk of its development [65]. Nevertheless, the effect of antioxidants in
the AD depends on various factors, such as on the quantity of antioxidants consumed, on the
synergic activity of other molecules and on the type of antioxidant.
Antioxidants are a potential medication for the therapy and prophylaxis of diseases
caused by oxidative stress [1]. But before a substrate can be used as a medication several
parameters have to be considered, in particular its bioavailability, a most important
parameter that is defined as the rate and extent to which the active ingredient or therapeutic
moiety is absorbed from a drug product and becomes available at the site of drug action [66].
The bioavailability depends on various factors, as the stability, the permeability and the
solubility [66]. It is also important to know pharmacokinetic parameters, such as the
absorption and the distribution of the drug. Although the solubility and the permeability are
the most important parameters [66], in the oral bioavailability, the stability is also important
because if a drug is not stable it will not be able to reach the systemic circulation [67]. For
antioxidants, due to their low bioavailability, a high dose of the antioxidant is needed, which
inherently increases the cost of the medication [66]. The solubility of antioxidants in gastric
fluids depends on their structure and properties and may be very diverse. The stability of
antioxidants depends on various parameters, such as the solvent or biological medium, pH,
temperature and the presence of substances with which they can interact.
The permeability through the gastrointestinal tract is also a very important factor for
the bioavailability [68], because it describes the ability of the drug to pass through the
gastrointestinal membrane to the systemic circulation. The antioxidants that present a low
solubility have also a low bioavailability. And for those that are highly soluble, the activity
can be prolonged or the release can be sustained, modulating the drug [69].
13
Many antioxidants are not readily accessible for oral administration, since they do not
reach the systemic circulation, due to their low values of solubility, stability and
permeability. To use antioxidants for the therapy of degenerative diseases, they have to
reach the brain and therefore they must penetrate the blood brain barrier [70]. This latter
requirement is also important in other diseases, as cancer, since the antioxidants have to
reach the tumor; if the antioxidant is not stable enough to reach the tumor it has no effect.
The formulation of a medicament depends on its characteristics and the routes of
administration. The oral route is the preferred by the patient and different forms are
available: capsules and in liquid or tablet forms [71].
For drugs with low bioavailability the delivery systems have to be improved. Inclusion
complexes of antioxidants, like those formed with cyclodextrins, that are hydrophilic in the
outer side and hydrophobic in the inner side, can improve the solubility and the stability of
antioxidants [71]. Bioavailability may also be increased by chemically modifying the drug,
but also affect its activity [71].
Recently, novel delivery systems for drugs with low oral bioavailability were
developed, that are able to deliver the antioxidants without changing their properties and
activity. The self-emulsifying drug delivery system is used for lipophilic compounds with
low solubility and low permeability, like the coenzyme Q10 [72]. The lipossomes can also
be used as a drug delivery system due to its biocompatibility; they have both a hydrophilic
and a hydrophobic part [71]. Microparticles and nanoparticles composed of polymers may
form a matrix that encapsulates the drug and are able to sustain it until the desired releasing
[71].
1.2.4. Cosmetic applications
As mentioned before, oxidative stress causes conditions in our organism that are
associated with aging. Aging is also an important factor in the cosmetic industry, because of
the alterations of the skin and, nowadays, there are a wide range of anti-aging products to
combat these alterations, and antioxidants are among them [73].
For cosmetic applications it is also crucial that the products used are stable and
odorless. Oxidation can modify the smell of a given product and the use of antioxidants can
protect the products from oxidation.
14
The skin is at all-time exposed to a variety of external agents, like the ultraviolet (UV)
rays that can generate free radicals and cause damages to the skin cells, DNA, proteins, and
can also destabilize the membranes of keratinocytes.
The exposure of the skin to UV radiation can induce the oxidative stress and causes
cell damages. The UVB rays (with wavelengths between 290 and 320 nm) are absorbed by
epidermal chromophores and can cause molecular damages and produce ROS [74], which
further causes DNA damages. UVA rays (with wavelengths between 320 and 400 nm) can
penetrate the dermis more profoundly and increase the generation of ROS. These UVA and
UVB rays can also activate a great variety of transcription factors in skin cells, one of this
is the NF-kB, a transcription factor, that is involved in inflammation and in the response of
the cell to stress [75]. The NF-kB increases the production of enzymes that degrade collagen
and elastin, the matrix metalloproteinases (MMPs).
The exposure of the skin to UV radiation can lead to a variety of skin disorders,
inflammation and photo aging of the skin [2,76]. The photo aging of the skin is a cause of
the appearance of wrinkles, the loss of elasticity and also increases the fragility of the skin.
It was proved by Brosche and Platt [77] that the utilization of borage oil, containing
antioxidants, can lead to a skin barrier in elderly persons, decreasing so the loss of water
through the skin. The supplementation with antioxidants, like vitamin E, vitamin C, and
carotenoids can protect the skin against UV radiation [78].
15
1.4. Extraction of antioxidants
Prior to the use of antioxidants in specific applications of the pharmaceutical and the
cosmetic industries, they need to be either synthesized or extracted from natural matrices.
Few reports can be found on the synthesis of phenolic compounds [79–85].
Gallic acid was synthesized by Cu2+-mediated oxidation of 3-dehydroshikimic acid,
with the highest yield of (74 %) being achieved with the catalyst Cu(OAc)2 [81]. A
biotechnological alternative was also made, synthesizing gallic acid from glucose with
microbial catalysis [81]. For this synthesis, E. coli KL7/pSK6.161 that was cultivated for 48
h in a fermentation reactor under glucose-rich and nitrogen-rich culture conditions, was used
[81]. The yield obtained was however lower than with the synthesis by Cu2+-mediated
oxidation of 3-dehydroshikimic acid.
Vanillic acid can be synthesized by microbial catalysis using suspensions of cells at
high density under hypersaline conditions [85]. For the synthesis of vanillic acid, Halomonas
elongata DSM 2581T were grown on ferulic acid to convert the ferulic acid into vanillic
acid. In this study [85], the highest yield (82%) was obtained after 10 h and using 10 mM of
ferulic acid and 5 g/L of biomass in saline-phosphate buffer. Another example for the
synthesis of vanillic acid by microbial catalysis is the bioconversion of ferulic acid into
vanillic acid using a Vanillate-Negative Mutant of Pseudomonas fluorescens Strain BF13
[84]. These microorganisms were grown on medium containing glucose as the carbon source
[84]. The maximal concentration obtained was 0.11 mg/mL of vanillic acid [84].
The production of vanillin by microorganisms is commonly made using lignin,
eugenol, isoeugenol and ferulic acid as substrates [80]. The production of vanillin using
ferulic acid as substrate can be done with Pseudomonas fluorescens [83]. In this work [83],
the vanillin dehydrogenase-encoding gene of the Pseudomonas fluorescens BF13 was
inactivated and the highest yield obtained was 1.28 g/L. The production of vanillin using
isoeugenol can be made, for example, with a Bacillus pumilus strain [82]. The highest yield
obtained was from 10 g/L of isoeugenol (added 30 h after incubation) that was converted to
3.75 g/L vanillin in 150 h, and that gives a yield of 40.5% [82].
The syringic acid can be produced, for instance, by a mycelia-derived enzyme, that
degrades lignin and produces syringic acid in the solid medium of cultured L. edodes mycelia
[79].
16
The processes of synthesis are expensive and lagged behind the isolation from natural
sources, particularly from industrial by-products, that are attracting an increasing interest.
The isolation process of antioxidants from their natural sources involves two major
steps: 1) their extraction from natural sources (biomass); 2) their re-concentration and/or
purification. The first step frequently uses a solid-liquid extraction to obtain the antioxidants
from their solid source into a liquid solution [86]. The second step concentrates and purifies
the antioxidant from the extracts using liquid-liquid extraction methods. In these processes,
some important parameters have to be considered, namely efficiency, time-consumption,
economical as well as environmental costs. Hence, the extraction time should be as low as
possible but with the greatest extraction efficiency. It must also be taken into consideration
that the solvent should not be malignant for the environment and the amount used in the
extraction process should be kept to a minimum.
1.4.1. Solid-liquid extraction
The conventional methods, such as maceration, percolation and soxhlet extraction,
have usually low efficiency values and are time consuming. They may be deleterious to the
environment due to the utilization of a great quantity of organic solvents. Another
disadvantage of using soxhlet extraction is the relatively high temperature imposed to the
compounds to be extracted, that may cause damage to the thermo labile ones [4]. In the last
few years, other techniques were developed for the substitution of the conventional ones,
such as the supercritical fluid extraction (SFE), the accelerated solvent extraction (ASE), the
ultra-sound assisted extraction (UAE) and the microwave assisted extraction (MAE).
1.4.1.1 Supercritical fluid extraction method (SFE)
The supercritical fluid extraction method (SFE) consists of using a supercritical fluid
as the extraction solvent to extract target substances from a natural source. Critical fluids are
compounds that are liquid at temperatures and pressures over their critical point. Normally,
at the critical point, the fluids show property evidences not only of a liquid but also of a
gaseous substance. Also, at the critical point, small variations of pressure or temperature can
17
drastically change the properties of the fluid. Increasing the density of the fluid increases the
solubility of the solute; because density increases with pressure, increasing the pressure
increases the solubility [87]. Water and carbon dioxide are mostly used in SFE due to their
advantageous properties, such as non-flammability and non-toxicity, as well as their low cost
[88]. This technique has been largely employed in pharmaceutical and food applications
[87]. Supercritical fluids, as CO2, are not able to extract substances that are more polar than
themselves and so they are not suitable for extracting polar substances, such as polyphenols.
To increase the solubility of polar substances in the fluid, co-solvents, such as methanol or
ethanol, may be used [89]. However, the addition of co-solvents increases the time required
for the extraction as well as the operative costs since they have to be further removed. SFE
was already used to extract phenolic acids - gallic acid, syringic acid and vanillic acid - from
grape bagasse and Pisco residues, using supercritical CO2 and the ethanol as co-solvent [90].
1.4.1.2. Accelerated solvent extraction (ASE)
Accelerated solvent extraction (ASE) usea high temperatures and pressures for the
extraction of target compounds from solid matrices while employing liquid solvents [91].
The increase in the temperature of the extraction causes an increase in the solubility as well
as a decrease in the viscosity and the surface tension of the solvent, and therefore leads to
improved efficiencies of extraction. This method takes less time than conventional methods
and a large range of solvents can be used [91]. For those solutes that cannot be exposed to
high temperatures, high pressure values are employed in the extraction procedure. However,
this technique presents some major drawbacks, such as the high solvent consumption and
the use of expensive equipment. ASE was used in the extraction of phenolic compounds -
vanillic acid, gallic acid and syringic acid - from perennial herbs (P. alsaticum and P.
cervaria) [92].
1.4.1.3. Ultra-sound assisted extraction (UAE)
The ultra-sound assisted extraction (UAE) use ultra-sound waves to enhance the
extraction of solutes [93] from a solid matrix. The ultra-sound waves pass through the liquid
18
solvent reaching the solid; the waves facilitate the release of the solute and the entry of the
solvent into the solid to solvate the extract, by applying mechanical, physical and chemical
forces. This method is not as expensive as the methods described previously, because it does
not need complex instrumentation. It was applied in the extraction of gallic acid, vanillic
acid and syringic acid from perennial herbs, namely from P. alsaticum and P. cervaria [92].
In general, the ASE methods lead to better extraction efficiencies than the UAE [92].
1.4.1.4. Microwave assisted extraction (MAE)
Microwave assisted extraction (MAE) uses microwaves, i.e., electromagnetic non
ionizing energy for facilitating the partition of the solute from the sample to the solvent [94];
the microwaves are absorbed and converted into heat. This method does not require much
time neither involves high solvent consumption. There are two types of MAE, the closed and
the open vessel systems [95]. In the closed vessel system the solvent and the sample are
contained in closed vessels and the temperature and pressure are controlled. Because the
vessels are closed, the temperature can be increased to a value higher than the boiling point
of the solvent, and so, the efficiency is increased and the time for the procedure is reduced.
In the open systems, the solvent and sample are in an open vessel and so the temperature is
not controlled while operating at ambient pressure. The MAE method was used for the
extraction of antioxidants, such as gallic acid and syringic acid from raspberries [96].
Vanillin was also extracted using MAE from pods of Vanilla planifólia [97]. Comparing
with UAE, MAE usually leads to improved extractions of vanillin [97].
1.4.2. Comparison of solid-liquid extraction methods for gallic, syringic, and
vanillic acids and vanillin
In this section the solid-liquid extraction methods for antioxidants will be discussed in
more detail although some of them were already highlighted before.
For the extraction of gallic acid some methods are usually applied, like supercritical
fluid extraction (SFE) [90], accelerated solvent extraction (ASE) [92], ultra-sound assisted
extraction (UAE) [92] and microwave assisted extraction (MAE) [96]. With the SFE method
19
[90], using the critical fluid CO2 containing 10% (w/w) of ethanol at 40 °C, some residues
of phenolic acids were extracted from grape bagasse from Pisco, namely syringic, vanillic,
gallic, p-hydroxybenzoic, protocatechuic and p-coumaric acids, as well as quercetin with
global extraction yields of 5.5% at 20 MPa and 5.6% at 35 MPa. The concentration of gallic
acid obtained by this method was 4.48 g/kg of extract [90].
With the ASE method [92], at 100 ºC, and repeated for five times, the gallic acid was
obtained in the concentration of 9.85 g/kg of extract from the fruits of P. alsaticum and of
9.25 g/kg from the fruits of P. cervaria. In the same study [92], the gallic acid was also
extracted with the UAE method with yields of 8.54 g/kg from the fruits of P. alsaticum and
of 8.37 g/kg from P. cervaria. Gallic acid was also extracted from raspberry by MAE using
ethanol [96] and it was obtained at a concentration of 0.63 g/kg of dry weight of extract and
at 37.91 g/kg of dry weight of extract considering the total phenolics content.
The extraction of vanillic acid from its natural sources can be performed using, for
example, SFE [90], ASE [92] or UAE [92]. Vanillic acid was extracted from grape bagasse
from Pisco with SFE [90] using supercritical CO2 containing 10% (w/w) of ethanol at 40 °C.
The concentration of vanillic acid obtained was 4.3 g/kg of extract [90]. Vanillic acid was
also extracted with the ASE method [92], at 100 ºC, repeated for five times, and the vanillic
acid concentration obtained was of 4.34 g/kg of extract from the fruits of P. alsaticum and
of 4.83 g/kg of extract from the fruits of P. cervaria. In the same study [92] the vanillic acid
was also extracted with the UAE technique and it was obtained 3.41 g/kg of extract from the
fruits of P. alsaticum and 2.21 g/kg of extract from the fruits of P. cervaria.
The extraction of vanillin from its natural sources was already attained by maceration,
percolation and soxhlet methods [28]. However, nowadays, other more efficient methods are
used, like MAE [97] and UAE [97]. The vanillin was extracted from pods of Vanilla
planifólia with UAE [97] using different solvents and the highest concentration, of 9.9 g/kg
of plant material, was obtained using dehydrated ethanol as solvent. In the same study,
vanillin was also extracted using MAE [97]. Several solvents were also tested and the highest
concentration (18.6 g/kg of plant material) was again obtained with dehydrated ethanol [97].
Alike the other compounds previously described, syringic acid can also be extracted
from its natural sources using several extraction techniques, like SFE [90], MAE [96], UAE
[92] and ASE [92]. With ASE [92], at 100 ºC and repeated for five times, the syringic acid
was obtained in the concentration of 3.77 g/kg of extract from the fruits of P. alsaticum. In
20
the same study [92], the syringic acid was also extracted by UAE and it was obtained 1.99
g/kg of extract from the fruits of P. alsaticum and was not detectable in the fruits of P.
cervaria. With SFE approaches [90], using the critical fluid carbon dioxide and containing
10% (w/w) of ethanol at 40 °C, syringic acid was extracted from grape bagasse from Pisco.
The concentration of syringic acid obtained by this method was 10.78 g/kg of extract [90].
The syringic acid was also extracted from raspberry by MAE using ethanol and it was
obtained 2.17 g/kg of dry weight of extract [96].
Considering the studies presented in this section, it is shown that the highest
concentrations for the extraction of the four antioxidants was not obtained with the same
extraction method. For the gallic acid and the vanillic acid the best results were obtained
with the ASE [92], whereas for vanillin and syringic acid it was with the MAE [97] and SFE
[90], respectively. MAE is a good alternative to the other methods presented due to the low
time and solvent consumption [94]; nevertheless, only for the vanillin it was obtained a good
result. ASE that shows good extraction results for the gallic and vanillic acid have the
advantage that it do not need much time and many solvents can be used, but this method
provides a high solvent consumption and the equipment is very expensive [91]. So, even
though the SFE does only show the best extraction for the syringic acid this method is the
method that shows the best properties combined with good extraction yields. For gallic acid
and vanillic acid it shows a considerable extraction of 4.48 g/kg of extract and 4.3 g/kg of
extract, respectively [90]. Only for the vanillin it was not found a study employing SFE.
Additionally, this method has advantages, because of the use of H2O and CO2 as solvents,
which are non-flammable, non-toxic and are of low cost [88].
1.5. Liquid-liquid extraction
After the extraction of molecules from their natural sources, a solution of the extract
and some contaminants or undesired compounds is usually obtained. Then, it is necessary to
treat the solution so as to obtain the target molecules in high concentration and with high
purity level. This purification step is generally performed using chromatographic techniques
and liquid-liquid extraction (LLE) methods, the first approach requiring more sophisticated
equipment [98]. The LLE is an inexpensive method since it involves the use of organic
solvents, but it requires long extraction times and temperatures giving rise to possible extract
21
degradation. Moreover, environmental concerns may be at stake from the use of organic
solvents.
The most conventional liquid-liquid system combines water with an immiscible
compound, namely a volatile organic compound (VOC). The targeted molecule partitions
preferentially into one of the immiscible phases, according to the relative affinities for both
phases. This method is fast, simple, economic and also very efficient [6]. Nevertheless, most
of the used VOCs have disadvantages: they are toxic, flammable and volatile [5], so, efforts
were made to replace these harmful solvents by benign ones. An important breakthrough,
particularly for the extraction of biomolecules, was having aqueous solutions replacing
organic solvents, the aqueous biphasic systems (ABS). These systems use a combination of
two aqueous solutions of polymers, salts or a polymer and a salt, to replace the conventional
VOCs [6] with inherent advantages in biocompatibility and environmental benignity. ABS
will be described in more detail in the next section.
22
1.5.1. Aqueous Biphasic Systems (ABS)
In 1958, Albertsson introduced a novel liquid-liquid extraction method to replace the
widely used VOCs, namely the aqueous biphasic systems (ABS) [6]. In this liquid-liquid
extraction approach, two immiscible aqueous solutions are used. ABS are formed by two
non-compatible solutes, such as salt-salt, polymer-salt or polymer-polymer combinations,
although both are water soluble. One of the phases is enriched in one of the solutes and the
other in the other solute. Since both phases are mostly composed of water, this method may
be advantageously used to extract or purify biomolecules, such as proteins, cell organelles
and nucleic acids due to its biocompatibility [99]. The solute concentration at which the two
phases are formed depends not only on the nature of the components but also on other
variables such as temperature, pH or ionic strength [6,100].
Since the 80s, ABS have been mostly used combining two polymers or a polymer and
a salt [101]. The salt has the advantage of lowering the viscosity and also the cost of the
overall system and hence polymer-salt ABS were preferred to polymer-polymer systems
[101]. However, salt solutions may have some inconveniences in the separation process of
biomolecules and other substances, due to the eventually high charged ions and the
environmental concerns some may pose.
Before ABS can be used as an extraction technique the concentrations of the
components at which they form two phases, that is, the solubility curve or binodal curve
must be determined. In Figure 1.5, an example of the phase diagram of an ABS composed
of poly(ethylene glycol) (PEG) and a salt solution is shown. The region above the binodal
curve corresponds to compositions where two phases are observed; under the binodal curve
is located the compositions at which the system is miscible, and so, a homogeneous solution
is found. Figure 1.5 also depicts three biphasic mixtures (X, Y and Z) with different
concentrations of each component and different volumes ratio. Nevertheless, because they
are placed along the same tie-line (TL), they all have the same top and bottom phase
compositions with only the volumetric ratio being different [102].
23
Figure 1.5- Phase diagram of an ABS composed of PEG and a salt aqueous solution [102].
The tie-line length (TLL) is a parameter related to the mass and the differences in the
compositions of the phases. The units of the TLL and of the composition are the same.
In the representation of Figure 1.5, the water concentration is omitted but it might be
explicitly represented using a triangular phase diagram, as shown in Figure 1.6. In this
diagram, the region under the curve is the biphasic region and the region over the curve is
the monophasic region. In this example a system composed of an ionic liquid (IL), which is
generally a salt that is liquid at temperatures below 100 °C, an inorganic salt and water is
depicted, but a similar diagram would be obtained for the systems composed of ILs and
polymers, having the inorganic salt replaced by the polymer.
24
Figure 1.6- Ternary phase diagram of an ABS composed by an ionic liquid and an inorganic salt aqueous
solution [101].
Hence, to experimentally characterize an ABS system, the respective phase diagram,
that is, the solubility curve, the tie-lines and tie-line lengths, must be determined.
ABS composed of different solutes have been used to extract antioxidants from several
matrices. ABS composed of an alcohol and a salt were used to extract vanillin from pudding
powder samples [103]. In this study, also the L-ascorbic acid was extracted with success
[103]. The alcohols that were studied include methanol, ethanol, 1-propanol and 2-propanol
in combination with aqueous solutions of the salts K2HPO4/KH2PO4, K3PO4 and K2HPO4.
It was observed that vanillin partitions preferentially into the alcohol-rich phase, while L-
ascorbic acid migrates preferentially to the salt-rich phase [103].
Another example is the work of Hasmann et al. [104] that have shown that
antioxidants can be removed from a hydrolysate of rice straw with ABS composed of two
thermoseparating copolymers, ethylene oxide and propylene oxide. The systems were
studied using polymers of different molecular weights (1,100 g.mol−1, 2,000 g.mol−1 and
2,800 g.mol−1) and different percentages of the copolymers (5 wt. % to 50 wt. %). Variable
extraction efficiencies (EE%), ranging from 6 % to 80 %, were obtained for ferulic acid,
syringic acid, furfural, vanillin and syringaldehyde, depending on the copolymer used [104].
25
The highest EE% of 80 % was obtained when using the systems composed of 35 wt. % of
the copolymer of 2,000 g.mol−1 at 25 °C [104]. It was also observed that for the systems
composed of 50 wt. % of the copolymer with the highest molecular weight the extraction
could not be performed because at this percentage the system becomes highly viscous [104].
1.5.2. Ionic liquids (ILs)
ILs are salts with a melting temperature below 100 °C, and many of them below room
temperature - the room temperature ionic liquids (RTILs). They are composed of an organic
cation and an organic or inorganic anion. ILs have low melting points due to the large size
of one or both of the ions (cation and anion), the low symmetry, the weak intermolecular
interactions and the even distribution of charge at their ions. The most common IL cations
are shown in Figure 1.7. For the anion, either organic or inorganic anions, such as
dicyanamide, acetate, trifluoroacetate, trifluoromethylsulfate, bromide, chloride, nitrate,
perchlorate, chloroaluminate, tetrafluoroborate, or hexafluorophosphate, have been used
[105]. Some examples of IL anions are also shown in Figure 1.7.
26
Figure 1.7- Chemical structure of common IL cations and anions.
Ethylammonium nitrate ([CH3CH2NH3][NO3]), with the melting point of 13-14 °C,
was the first IL synthesized, by Paul Walden in 1914, when the author was testing new
explosives for the substitution of nitroglycerine [106]. But at the time, no importance was
given to that discovery. Only after 20 years, in 1934, Charles Graenacher patented, for the
first time, an industrial application of ILs for dissolving cellulose [107]. In 1948, Hurley and
Wier [108] developed ILs with chloroaluminate ions for the electrodeposition of aluminium
at low temperatures. In 1986, ILs were proposed as solvents for organic synthesis by Fry and
Pienta [109] and Boon et al. [110]. When the first air- and moisture-stable imidazolium ILs
were developed in 1992 [111], the interest for the ILs as alternative solvents largely
increased. Wilkes and Zaworotko [111] synthesized stable RTILs containing weakly
complexing anions, such as [BF4]-. Since then, the interest in studying ILs has been
27
increasing and, consequently, the number of publications increased exponentially as it can
be seen in Figure 1.8, where the increase in the number of published articles regarding ILs
per year from 1991 to 2013 is shown.
Figure 1.8- Number of published articles regarding ILs per year (in the last 20 years). Information taken
from ISI Web of Knowledge in 17th January 2014.
ILs are called “designer solvents” due to the possibility of adjusting their chemical and
physical properties by changing the cations and anions, such as the length of the alkyl chain
and addition of functionalized groups. So, properties such as melting point, viscosity, water-
miscibility, density, acid/base character, and coordinating ability may be tuned according to
specific needs [112,113].
They are also called “green solvents” because of their low toxicity, low vapor pressure,
low volatilities and non-flammability that make them more benign for the environment.
They also have wide liquid ranges, high thermal and chemical stabilities and high dissolution
capacity for various solutes [114,115]. The ionic interactions in ILs make them miscible in
polar substances and, on the other hand, their solubility in less polar substances may be
adjusted by the choice of the nature of the cation and by varying its alkyl side chains.
0
2000
4000
6000
8000
10000
12000
14000
16000
1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013
nu
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er o
f p
ub
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ed
arti
cle
s
Year
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Initial reports on the utilization of RTILs in chromatography and in extraction
processes, as well as their use for electroanalytical purposes, began to appear in the late
1990’s [116]. RTILs are also used in sensing, spectrometry, separation, biocatalysis, organic
synthesis and extraction of biomolecules due to their non-denaturation properties [116].
In 2003, Rogers and co-workers [117] demonstrated, for the first time, the possibility
of forming ABS with inorganic salts and ionic liquids (ILs). IL-based ABS are a good
alternative to conventional separation techniques due to the advantageous properties of ILs:
non-volatility, that is the most important, low flammability, chemical and thermal inertness,
and moderate viscosity [114,115]. Moreover, one of the most outstanding benefits of IL-
based ABS is that the properties of the coexisting phases may be changed by a proper choice
of the ILs, since the anion or the cation nature and/or their characteristics, allows the
adjustment of their properties to suit the needs of a target study [112,113].
Imidazolium-based ILs are the most extensively studied ILs regarding their use to form
ABS [101]. Their properties can be significantly changed, for example, by modifying the
alkyl chain length we can obtain imidazolium-based ILs completely miscible in water to
nearly immiscible ones. Nevertheless the imidazolium-based ILs have some disadvantages,
the most important one is the ecotoxicity that increases with the increase of the alkyl chain
length [118].
The phosphonium-based ILs consist of a quaternary phosphonium cation; they are
chemically and thermally more stable than imidazolium- and ammonium-based ILs [7].
Phosphonium-based ILs were already investigated as phase-forming components of ABS
and, in general, they are more prone to undergo liquid-liquid demixing when compared to
their imidazolium-based counterparts [101]. Also, they are generally less dense than water
which is advantageous in downstream processing [7].
The cholinium-based ILs consist on a quaternary ammonium cation, and in particular
the N,N,N-trimethylethanolammonium cation. They have received an increasing interest in
the last years due to their improved properties when compared to other ILs [8]; they are not
as expensive as the conventionally used ILs [119]; cholinium-based ILs can be very easily
regenerated and are also thermally stable. In addition, they are more benign and
biocompatible than other ILs. In fact, choline is a water soluble vitamin that is very important
in assuring good health conditions [120]. These ILs also have antimicrobial and anti-
electrostatic properties, and these properties lead to their use in a variety of applications
29
substituting other ILs [8]. ABS composed of cholinium-based ILs were also reported, either
combined with inorganic salts or polymers [121,122].
There are several studies in the literature that report the use of IL-based ABS for the
extraction of phenolic compounds [103,123–125], and in the next paragraphs their main
results are described.
Vanillin was extracted with ABS constituted by ILs and the salt K3PO4 [124]. In this
study, the partition coefficients (K) of vanillin using different ILs were determined. The
effect of the IL cation on the K values was ascertained using imidazolium-based ILs: (1-
ethyl-3-methylimidazolium chloride ([C2mim]Cl), 1-butyl-3-methylimidazolium chloride
([C4mim]Cl), 1-hexyl-3-methylimidazolium chloride ([C6mim]Cl), 1-heptyl-3-
methylimidazolium chloride ([C7mim]Cl), 1-decyl-3-methylimidazolium chloride
([C10mim]Cl), 1-allyl-3-methylimidazolium chloride ([amim]Cl), 1-benzyl-3-
methylimidazolium chloride ([C7H7mim]Cl) and 1-hydroxyethyl-3-methylimidazolium
chloride ([OHC2mim]Cl)). It was found out that the cation has a significant influence on the
partition; the distribution coefficient increases when the alkyl chain length increases from
[C2mim]Cl to [C6mim]Cl, but the trend inverts, the coefficient decreases, with the increase
of the alkyl chain from [C7mim]Cl to [C10mim]Cl. For the other ILs the partition coefficient
for the extraction of vanillin decreases in the sequence: [C7H7mim]Cl > [amim]Cl >
[OHC2mim]Cl. The effect of the anion on K was also studied; ILs composed of the cation
[C4mim]+ and different anions ([C4mim]Cl, 1-butyl-3-methylimidazolium bromide
([C4mim]Br); 1-butyl-3-methylimidazolium methanesulfonate ([C4mim][CH3SO3]); 1-
butyl-3-methylimidazolium acetate ([C4mim][CH3CO2]), 1-butyl-3-methylimidazolium
methylsulfate ([C4mim][CH3SO4]), 1-butyl-3-methyl-imidazolium
trifluoromethanesulfonate ([C4mim][CF3SO3]) and 1-butyl-3-methylimidazolium
dicyanamide ([C4mim][N(CN)2])) were investigated. Also the IL anion has a significant
effect on the vanillin partitioning and follows the sequence: [C4mim]Cl > [C4mim][N(CN)2]
> [C4mim]Br > [C4mim][CH3SO4] > [C4mim][CF3SO3] > [C4mim][CH3SO3] ≈
[C4mim][CH3CO2]. In summary, for all the ILs investigated, vanillin preferentially
partitioned for the IL-rich phase [124]. The IL cation and anion structure, the temperature of
equilibrium and the concentration of the solute were found to affect the partition of the
antioxidant [124]. The highest K value, 49.59, was obtained for the system composed
[C6mim]Cl + K3PO4 at 25 °C [124].
30
Gallic acid was extracted using ABS based on ILs and different inorganic salts, namely
Na2SO4, K2HPO4/KH2PO4 and K3PO4 [123]. The effect of the inorganic salts on the partition
coefficients for the extraction of gallic acid was studied. Different ILs were also studied: 1-
ethyl-3-methylimidazolium trifluoromethanesulfonate ([C2mim][CF3SO3]), [C4mim]Br,
[C4mim][CH3SO4], 1-butyl-3-methylimidazolium ethylsulfate ([C4mim][C2H5SO4]),
[C4mim][CF3SO3], [C4mim][N(CN)2], [C7mim]Cl, 1-octyl-3-methylimidazolium chloride
([C8mim]Cl) and 1-butyl-3-methylimidazolium octylsulfate ([C4mim][OctSO4]). For all
those ILs with the salt K3PO4, the K values decrease in the following sequence: [C7mim]Cl
> [C8mim]Cl > [C4mim][CH3SO4] ≈ [C4mim]Br > [C2mim][CF3SO3] ≈ [C4mim][N(CN)2]
> [C4mim][OctSO4] > [C4mim][CF3SO3]. For the system based on the buffer
K2HPO4/KH2PO4 the sequence decrease in the following order: [C4mim]Br > [C8mim]Cl >
[C4mim][CH3SO4] > [C7mim]Cl > [C4mim][N(CN)2] > [C4mim][CF3SO3] >
[C2mim][CF3SO3] and for the Na2SO4-based ABS it decreases in the following order:
[C4mim][C2H5SO4] > [C4mim][CH3SO4] > [C4mim]Br ≈ [C4mim][CF3SO3] ≈ [C7mim]Cl ≈
[C4mim][N(CN)2] > [C2mim][CF3SO3] > [C8mim]Cl > [C4mim][OctSO4]. Regarding the
salt used, and for most ILs, the partition coefficient decrease in the order: Na2SO4 >
K2HPO4/KH2PO4 > K3PO4. For the most of the systems studied the gallic acid partitioned
preferentially to the IL-rich phase; the best recovery was obtained with the Na2SO4-based
systems at the IL-rich phase with extraction efficiencies around 99% [123].
1.5.3. Polymers
As mentioned before, the possibility of forming ABS with a polymer and an inorganic
salt dates back in 1986. For ABS composed of two polymers, the mostly used polymers are
the poly(ethylene glycol) (PEG) and dextran because they are stable and non-toxic [126].
These are examples of two hydrophilic polymers, a synthetic (PEG) and a natural one
(dextran), but a few more may be found, namely poly(ethylene oxide) (PEO), poly(propylene
glycol) (PPG), chitosan, poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) are
examples of synthetic polymers, while dextran and maltodextrin are natural polysaccharides.
PEG and PEO have the same monomer unit; while PEO designates the polymer with
molecular weights above 20,000 g.mol-1, PEG is the designation when the molecular weight
is below 20,000 g.mol-1; both have a hydroxyl group at one end and a hydrogen atom at the
31
other. The monomer of PVA is isomeric with that of PEG and PEO. PPG although bearing
a structure similar to PEG and PEO has a more hydrophobic character. The monomer
structures of PEG/PEO, PPG and PVA are presented in Figure 1.9.
(i) (ii) (iii)
Figure 1.9-Molecular structures of (i) PEG/PEO, (ii) PPG and (iii) PVA.
Polymer-polymer ABS contains two uncharged phases whose polarities depend on the
quantity of water present in each phase, which, in turn, depends on the relative
hydrophilicities of both polymers. In polymer-salt ABS, there is a polymer-rich and less
hydrophilic phase and a more hydrophilic phase enriched in the charged salt. To adjust the
polarity of the phases according to the target molecule, the salt identity or the polymer may
be changed. As mentioned before, the replacement of a polymer by a salt has several
advantages, namely reducing the viscosity allowing a faster separation, but may pose
solubility inconveniences and environmental concerns. The replacement of the inorganic salt
by an ionic liquid can prevent problems associated with the crystallization of the common
inorganic salts that have high melting points, due to the low melting point of ILs, while
maintaining the viscosity of the phases low [101]. Additionally, in the ionic-liquid-polymer
ABS the polarity of the phases can be finely controlled [127]. In spite of these promising
attributes, only a few works combining ILs and polymers were published [127–131] using
either PPG [129,130] or PEG [127,128,131].
Rebelo and co-workers [128] published a pioneering work on the effects of salting-in
and salting-out of ILs by polymers in aqueous solutions. The polymer used was PEG with
the molecular weight of 35,000 g.mol-1 and the authors concluded that the use of ILs and
inorganic salts can fine-tune the salting-in or the salting-out effects through PEG.
Comparing the results from Wu et al. [130] and Zafarani-Moattar et al. [129] obtained
with 1‐ethyl‐3‐methylimidazolium bromide ([C2mim]Br) and [C4mim]Br, it could be
inferred that the ILs with a shorter alkyl chain at the cation promote the easier formation of
ABS; the same can be observed for ABS based on PEG in the work of Freire et al. [131].
Polyvinyl pyrrolidone polyvinyl alcohol
32
This effect is due to the increased hydrophilicity of the ILs by decreasing the alkyl side chain
of the cation. However, when combining the same ILs with inorganic salts, the opposite
effect is observed; increasing the alkyl chain of the ILs cation, and so its hydrophobicity,
causes the ability to form ABS to increase [101].
In the IL-polymer ABS, not only the ionic liquids can be manipulated but also the
polymers, by changing the nature and the length of the polymeric chains. Wu et al. [130]
studied the ABS formation ability with PPG of 400 g.mol-1 and PPG of 1,000 g.mol-1 and
Freire et al. [131] with PEG of 1,000 g.mol-1, PEG of 2,000 g.mol-1, PEG 3,400 g.mol-1 and
PEG of 4.000 g.mol-1, both using [C4mim]Cl. Comparing both works [130,131] it is evident
that either for PEG as for PPG, increasing the molecular weight increases the ability to form
ABS. The decreased affinity for water of both polymers as their molecular weight increases
leads to an easier liquid-liquid demixing. Comparing the ABS based on PPG of 1.000 g.mol-
1 and PEG of 1.000 g.mol-1, it may be seen that PPG of 1.000 g.mol-1 has a higher ability to
form ABS than PEG of 1.000 g.mol-1 [130,131]. A possible explanation for this is that PPG,
having an additional methyl group compared to PEG, is more hydrophobic and so more
easily salted-out by the IL.
PEG-based ABS were used to study the extraction of gallic acid, vanillic acid and
syringic acid [132]. The phase diagrams of the systems composed of Na2SO4 and PEG of
different molecular weights (200 g.mol-1, 300 g.mol-1, 400 g.mol-1 and 600 g.mol-1) were
determined and it was found that increasing the molecular weight of the PEG increases the
ability of the system to form two phases [132]. The influence of the ILs added to these
systems was also investigated. The ILs studied were: 1-butyl-3-methylimidazolium
thiocyanate ([C4mim][SCN]), 1-butyl-3-methylimidazolium tosylate ([C4mim][Tos]),
[C4mim][N(CN)2], [C4mim][CH3CO2], [C4mim]Cl; 1-butyl-1-methylpiperidinium chloride
([C4mpip]Cl) and 1-butyl-1-methylpyrrolidinium chloride ([C4mpyr]Cl). For
[C4mim][N(CN)2]. The effect of the concentration of IL in the two-phase forming ability
was studied preparing ABS systems with the addition of 5 wt. %, 10 wt. % and no IL. It was
shown that increasing the amount of IL added increases the ability to form ABS [132]. The
influence of the anion was studied with the different imidazolium-based ILs and it was
demonstrated that the ability to form ABS increases according to the following sequence of
the anions: [CH3CO2]- ≈ Cl- < [Tos]- < [SCN]- ≈ [N(CN)2]
-. Finally the cation influence was
33
also investigated using the ILs with the anion Cl- and three different cations and the ability
increases in the sequence: [C4mim]+ < [C4mpyr]+ < [C4mpip]+ [132].
The partition coefficient (K) of the gallic acid using systems without IL decreases with
the decrease of the molecular weight of the PEG, only for PEG of 400 g.mol-1 the K is higher
than for PEG 600 g.mol-1 and only for this PEG the pH is significantly lower than for the
other molecular weights. The EE% was found to be approximately the same (94 %) for all
the PEGs, the value for PEG of 400 g.mol-1 being slightly higher (96 %). For all the systems,
gallic acid partitioned preferentially to the PEG-rich phase [132]. The addition of the IL
[C4mim]Cl was studied for the system composed of PEG of the lowest molecular weight.
The system compositions used for the comparison of the extraction of the three antioxidants
with and without IL were 23 wt. % PEG 300 g.mol-1 + 12 wt. % Na2SO4 and 23 wt. % PEG
300 g.mol-1 + 12 wt. % Na2SO4 + 5 wt. % [C4mim]Cl. The comparison shows that the
addition of the IL increases both K and EE% values of the three antioxidants; extraction
efficiencies of 98% (gallic acid) and 99% (vanillic acid and syringic acid) were obtained
[132].
As mentioned above, ABS based on ILs and polymers have not been the focus of many
studies and only a few polymers were characterized for their use in ABS. Yet, polymers have
a very good potential for ABS extraction procedures [129–131]. So, it is mandatory to
investigate the use of other polymers and to perform the adequate characterization of
polymer-based ABS.
34
35
2. IL + PVP + H2O ABS
36
37
2.1. Poly(vinyl pyrrolidone) (PVP)
PVP, also called polyvidone or povidone, is a synthetic polymer composed of
monomers of N-vinylpyrrolidone; the IUPAC name is 1-ethenylpyrrolidin-2-one. It was
patented by Walter Reppe in 1939 [133] and its molecular structure is represented in Figure
2.1.
Figure 2.1- Chemical structure of PVP.
PVP can be obtained in a wide range of molecular weights [134]. It is soluble in water
and in many solvents, such as methanol, ethanol, chloroform and glycerol, although the
solubility depends on the molecular weight; low-molecular weight PVPs are more soluble
in water than high-molecular weight PVPs [134]. The viscosity of aqueous solutions
increases with the increasing of the molecular weight of PVP, and decreases with increasing
the temperature [134]; however it is not much affected by electrolytes [135].
PVPs have a large variety of advantageous properties; they are physiologically
compatible, non-toxic and stable at high temperatures and at different pH values [134]. It
was used since World War II as a blood plasma substitute; however, low quantities of PVP
remain in the body when used in parenteral administration because it is not metabolized
(contrary wise to the oral administration). PVP use as a blood plasma substitute was
discontinued but it still has some pharmaceutical applications. It is used as a binder in many
pharmaceutical tablets because it simply passes through the body when taken orally. PVPs
are also employed in cosmetic and technical industries [136]. More than 140 papers between
1960 and 1990 describe the preparation of drugs in solid solution, dispersions using PVP
[137]. Many of the active substances have poor aqueous solubility due to which they have
limited bioavailability. An easy way of enhancing the bioavailability of an active substance
Polyvinyl pyrrolidone polyvinyl alcohol
38
is to improve its dissolution by adding solubilizing agents, such as the soluble PVP grades.
These soluble PVP grades are also useful for preparing solid solutions and dispersions
because of their good hydrophilization properties, universal solubility and ability to form
water soluble complexes. When added to iodine, PVP forms a complex called povidone-
iodine that possesses disinfectant properties. This complex is used in various products like
solutions, ointments, pessaries, liquid soaps and surgical scrubs [134].
The U.S. Food and Drug Administration (FDA) has approved PVP for many uses
[138] and it is generally considered safe. It has been used as a food additive and as a
stabilizer. It can also be used in the wine industry as a fining agent for white wines and some
beers [135]. PVP has adhesive and binding properties, it can form films and it has thickening
properties; examples of technical applications are adhesives, textile auxiliaries and
dispersing agents [135].
ABS composed of PVP and inorganic salts were already reported and applied in the
partitioning of ions [139]; PVP 10,000 g.mol-1 and the inorganic salt (NH4)2SO4 were the
components used in the extraction of the anion pertechnetate. Another example is the
partition of colloidal particles using two polymers: PVP 360,000 g.mol-1 and PEGs of
different molecular weights (400,000 g.mol-1, 200,000 g.mol-1 and 100,000 g.mol-1); it was
shown that the particles having an hydroxyl group at the surface migrate preferentially to the
PVP-rich phase and those with a carboxyl group have more affinity to the PEG-rich phase
[140]. PVP of 12,700 g.mol-1 was also shown to form ABS when combined with the
polysaccharide dextran (57,200 g.mol-1 ) [141]. In that report, the behavior of the binodal
curves upon addition of aqueous solutions of inorganic salts (KSCN and K2SO4) was also
studied and it was found that the ability to form ABS in presence (or not) of a salt follows
the sequence: K2SO4 > no salt added > KSCN [141]. In summary, the addition of a salt can
favor (K2SO4) or disfavor (KSCN) the ABS ability of this type of systems [141].
As mentioned before, the use of ABS based on ILs and polymers permits to adjust the
properties of the system according to the molecule that is going to be extracted. The
properties of the ILs can be adjusted by modifying the nature and the characteristics of their
ions. Also the properties of PVP may be modified by changing the number of monomers,
that is, the molecular weight of the polymer. Nevertheless, up to now, there are no reports
about ABS composed of PVP and ILs. In this study, PVP was combined with several IL
39
families to investigate their ABS forming ability aiming at their subsequent use as extraction
systems for antioxidants.
40
2.2 Experimental section
2.2.1. Chemicals
Poly(vinyl pyrrolidone) (PVP) was purchased from Sigma-Aldrich; three different
molecular weights are used in this work, namely 40,000 g.mol-1 (PVP40), 29,000 g.mol-1
(PVP29) and 10,000 g.mol-1 (PVP10).
The phosphonium-based ILs used were: tributyl(ethyl)phosphonium diethylphosphate
([P4442][Et2PO4]) > 95.0 wt. % pure, tetrabutylphosphonium bromide ([P4444]Br) > 96.0 wt.
% pure, tetrabutylphosphonium chloride ([P4444]Cl) > 96.0 wt. % pure,
triisobutyl(methyl)phosphonium tosylate ([Pi(444)1][Tos]) > 99.0 wt. % pure and
tributyl(methyl)phosphonium methylsulfate, ([P4441][CH3SO4]) > 98.6 wt. % pure. All ILs
were kindly supplied by Cytec Industries, Inc. The ILs that presented higher water contents,
[P4444]Br and [P4444]Cl, were dried under vacuum at moderate temperature (≈ 80 °C) and
constant stirring for a minimum of 72 h. After drying and before use, the purity of the ILs
was verified by 1H, 13C and 31P NMR.
The imidazolium-based ILs investigated were: 1-ethyl-3-methylimidazolium acetate
([C2mim][CH3CO2]) > 98.0 wt. % pure, 1-ethyl-3-methylimidazolium hydrogensulfate
([C2mim][HSO4]) > 98.0 wt. % pure, 1-ethyl-3-methylimidazolium dimethylphosphate
([C2mim][PO4(CH3)2]) > 98.0 wt. % pure, 1-butyl-3-methylimidazolium
trifluoromethanesulfonate ([C4mim][CF3SO3]) 99.0 wt. % pure, 1-butyl-3-
methylimidazolium thiocyanate ([C4mim][SCN]) > 98.0 wt. % pure, 1-butyl-3-
methylimidazolium tetrafluoroborate ([C4mim][BF4]) 99 wt. % pure, 1-butyl-3-
methylimidazolium dimethylphosphate ([C4mim][PO4(CH3)2]) > 98.0 wt. % pure, 1-butyl-
3-methylimidazolium tosylate ([C4mim][Tos]) 98.0 wt. % pure and 1-hexyl-3-
methylimidazolium dicyanamide ([C6mim][N(CN)2]) > 98.0 wt. % pure. All imidazolium-
based ILs were purchased from Iolitec.
The cholinium-based ILs used were: cholinium dihydrogenphosphate ([Ch][DHPh])
98 wt. % pure, cholinium dihydrogencitrate ([Ch][DHCit]) 98 wt. % pure and cholinium
acetate ([Ch][CH3CO2]) 99 wt. % pure, from Iolitec; cholinium bicarbonate ([Ch][Bic]) <
80 wt. % pure, cholinium bitartrate ([Ch][Bit]) 99 wt. % pure and cholinium chloride
([Ch]Cl) 99 wt. % pure, from Sigma-Aldrich. The following were synthesized in our
41
laboratory according to standard protocols [142,143]: cholinium propionate ([Ch][Prop])
and cholinium lactate ([Ch][Lac]). These two ILs were purified and dried for a minimum of
24 h at constant stirring, at 80 °C and under vacuum to reduce the volatile impurities. The
purity of these ILs was confirmed by 1H and 13C NMR spectra and found to be > 98 wt. %.
The protic ammonium-based ILs studied were: N,N-dimethyl-N-ethylammonium
acetate ([N1120][CH3CO2]) 98 wt. % pure, N,N-dimethyl-N-(N´,N´dimethylaminoethyl)
ammonium chloride ([N11[2(N110)]0]Cl) 97 wt. % pure and N,N-dimethyl-N-
(N´,N´dimethylaminoethyl) ammonium acetate ([N11[2(N110)]0][CH3CO2]) 98 wt. % pur). All
the protic ammonium-based ILs were purchased from Iolitec.
The molecular structures and the molecular weights of the studied ILs are presented in
Table 2.1.
The antioxidants used were: gallic acid > 99.5 wt. % pure from Merck; vanillin > 99
wt. % pure from Acros organics; vanillic acid > 97 wt. % pure from Aldrich; and syringic
acid > 98 wt. % pure from Alfa Aesar. The chemical structures of the antioxidants are
represented in Figure 1.2 in section 1.2.1.
Acetone > 99.5 wt. % pure from Chem-Lab was used for the polymer precipitation.
Buffer solutions of biphthalate - pH 4.00 (at 25 °C) and phosphate - pH 7.00 (at 25 °C)
used for pH calibration were purchased from Metrohm.
Water was distilled twice, passed by a reverse osmosis system and additionally treated
with a Milli-Q plus 185 water purification system.
42
Table 2.1- Cation an anion structure and molecular weights of the ILs studied.
IL Cation structure Anion
structure
Molecular weight /
(g.mol-1)
Phosphonium-based ILs
[P4444]Br
339.33
[P4444]Cl 294.88
[Pi(444)1][Tos]
388.54
[P4441][CH3SO4]
328.45
[P4442][Et2PO4]
384.47
Cholinium-based ILs
[Ch][DHPh]
201.16
[Ch]Cl 139.62
[Ch][CH3CO2]
163.21
[Ch][Bic]
165.19
[Ch][Bit]
253.25
[Ch][DHCit]
295.29
[Ch][Prop]
177.24
[Ch][Lac]
176.23
43
Imidazolium-based ILs
[C2mim][CH3CO2]
170.21
[C2mim][HSO4]
208.24
[C2mim][PO4(CH3)2]
236.21
[C4mim][CF3SO3]
288.29
[C4mim][SCN] 197.30
[C4mim][HSO4]
236.29
[C4mim][PO4(CH3)2]
264.14
[C4mim][Tos]
310.41
[C4mim][BF4]
226.02
[C6mim][N(CN)2]
234.32
Protic ammonium-based ILs
[N1120][CH3CO2]
133.19
[N11[2(N110)]0]Cl
167.68
[N11[2(N110)]0][CH3CO2]
177.26
44
2.2.2. Experimental procedure
2.2.2.1. Phase diagrams and tie-lines
The phase diagrams were determined by the cloud point titration method at (25 ± 1)
°C and atmospheric pressure. At first, aqueous solutions of PVP of low concentrations were
prepared (24 wt. % for PVP40 and 35 wt. % for PVP29), because of their high viscosity at
high concentrations. But when performing the determination of the phase diagrams, only
few points were obtained when using these solution concentration values for some ILs and
other systems did not even reach the concentrations at which they form two phases. It was
then attempted to use more concentrated solutions in the phase diagrams determination.
Aqueous solutions of PVP40 (at 46 wt. %), PVP29 (at 54 wt. %) and PVP10 (at 56 wt. %)
and concentrated solutions of phosphonium-based ILs at different concentrations (80% to
100%) were prepared. To obtain the phase diagrams, the IL solution was added drop-wise
to the PVP solution until the system becomes cloudy (biphasic region); then, water was drop-
wise added until a clear solution is obtained (monophasic region) – Figure 2.2. The process
was repeated, under constant stirring. The whole procedure of experimentally determine the
phase diagrams was made twice so as to obtain more points on the phase diagrams and to
ascertain on the reliability of the experimental values. For the region of the phase diagram
where the concentration of IL is high and the concentration of PVP is low, a slightly different
procedure was made. The cloud point titration method was also the method used, but, instead
of adding IL solution to the PVP solution, the PVP solution was added to the IL solution;
the concentrations of both solutions used were the same as those mentioned before. To
determine the ternary system compositions the weight of all added components was
measured within an uncertainty of ± 10-4 g.
45
Figure 2.2- Experimental determination of the phase diagrams. (A) Cloudy solution (biphasic region); (B)
Clear solution (monophasic region).
The experimental binodal curves were fitted using equation 1 [144],
[𝑃𝑉𝑃] = 𝐴𝑒𝑥𝑝[(𝐵[𝐼𝐿]0,5) − (𝐶[𝐼𝐿]3)]
where [PVP] and [IL] are respectively the PVP and IL weight percentages and A, B and C
are parameters obtained by the non-linear regression.
For the determination of the tie-lines (TLs) a fixed point within the biphasic region
was chosen for every system (close to 67 wt. % of IL and 13 wt. % of PVP); the required
amounts of PVP, IL and water were added, vigorously agitated until dissolution and then left
to equilibrate. To ensure the complete separation of the two phases they were left to rest for
at least 12 hours at 25 °C. After equilibration, they were centrifuged at 3500 rpm for 10
minutes to improve the separation of the two phases. These were carefully separated with
the aid of a pipette and both phases were weighted.
From the total mass of each phase, the TL composition can normally be estimated by
solving the following system of four equations (Equations 2 to 5) and four unknown values
([IL]IL, [IL]PVP, [PVP]IL and [PVP]PVP) [144],
[𝑃𝑉𝑃]𝐼𝐿 = 𝐴 ∗ exp[(𝐵 ∗ [𝐼𝐿]𝐼𝐿0.5) − (𝐶 ∗ [𝐼𝐿]𝐼𝐿
3 )]
[𝑃𝑉𝑃]𝑃𝑉𝑃 = 𝐴 ∗ 𝑒𝑥𝑝[(𝐵 ∗ [𝐼𝐿]𝑃𝑉𝑃0.5 ) − (𝐶 ∗ [𝐼𝐿]𝑃𝑉𝑃
3 )]
[𝑃𝑉𝑃]𝑃𝑉𝑃 =[𝑃𝑉𝑃]𝑀
𝛼− (
1 − 𝛼
𝛼) ∗ [𝑃𝑉𝑃]𝐼𝐿
[𝐼𝐿]𝑃𝑉𝑃 =[𝐼𝐿]𝑀
𝛼− (
1 − 𝛼
𝛼) ∗ [𝐼𝐿]𝐼𝐿
(1)
A B
(4)
(5)
(2)
(3)
46
where the subscripts IL, PVP and M represent the IL-rich top phase, the PVP-rich bottom
phase and the initial mixture, respectively. The parameter α is the ratio between the top
weight and the total weight of the mixture.
However, and although the previous equations were shown to be applicable in IL +
salt ABS [145,146], when trying to solve the system using the experimental data for IL +
PVP ABS, no convergence was observed; so it was not possible to obtain an estimate for the
concentrations of the IL and PVP in the top and bottom phases. Consequently, the
composition of each phase had to be experimentally determined. Since PVP is not soluble in
acetone, its content in each of the two phases was determined by precipitation with this
solvent. To optimize the procedure, preliminary assays were made using different
acetone/PVP proportions; solutions of PVP40 at 25 wt. % in water were prepared and
acetone was added in the following amounts of PVP-solution: acetone (w:w): 1:1, 1:2, 1:3
and 1:4. The solutions were then centrifuged at 3500 rpm for 30 min and the excess of
acetone was decanted; PVP was washed twice with acetone within the same conditions to
obtain a better estimate of the PVP weight; the precipitate was then dried at 70 ºC until
constant weight, and the dry weight was used to calculate the yield of the PVP precipitation.
It was found that optimal results for the precipitation of PVP are achieved when acetone is
added to PVP solutions in the amounts of (1:4, w:w) with recovery efficiencies of 98.61 %
– this value was obtained as the average of 3 individual precipitation assays. This procedure
was then followed to determine the PVP content at the ABS phases: acetone was added to a
known amount of each phase (1:4, w:w) and the mixture was centrifuged at 3500 rpm for 30
min. The acetone was removed firstly by decantation and then by evaporation as previously
described. Finally, the dried PVP was weighted.
The amount of water in each phase was determined also in the evaporation step. A
known amount of each phase was left at 70 °C until constant weight. Since neither PVP nor
IL evaporate under these conditions, water loss accounts for the observed weight difference.
The amount of IL was determined by subtracting PVP and water weights to the initial weight
of each aqueous phase. Duplicate assays were performed for each TL determination.
47
2.2.2.2. pH measurements
The pH values of both the IL-rich and PVP-rich phases were measured at (25 ± 1) °C
using a Mettler Toledo S47 SevenMultiTM dual meter pH/conductivity equipment with an
uncertainly of ± 0.02 pH units. The electrode was calibrated using buffer solutions of
biphthalate for pH=4.00 and phosphate for pH=7.00.
2.2.2.3. Partition of antioxidants
For the extraction of the antioxidants, aqueous solutions of gallic acid (1 g.dm-3),
vanillin (1 g.dm-3), vanillic acid (0.5 g.dm-3) and syringic acid (0.5 g.dm-3) were initially
prepared.
To study the partition of the antioxidants, a fixed composition in the biphasic region
was used: 67 wt. % of IL + 13 wt. % of PVP + 20 wt. % of antioxidant solution (Figure 2.3);
the solution was prepared and the mixture was vigorously agitated until completely
dissolution. To ensure their complete separation, the two phases were
left at rest at least for 12 hours at 25 °C; after that period they were
centrifuged at 3500 rpm for 10 min. The two phases were then
carefully separated and weighted, the pH of each phase was
determined and the quantification of each antioxidant at each phase
was performed. At least three independent samples were prepared and
analyzed for each system.
The antioxidant concentration was quantified in each phase by
UV-spectroscopy, carried out with a SHIMADZU UV-1800
spectrophotometer, at a wavelength of 262 nm for gallic acid, 274 nm
for syringic acid, 280 for vanillin and 292 nm for vanillic acid. The
calibration curves were previously determined for each antioxidant
and can be found in Appendix A. Blank control samples were always
prepared.
The partition coefficients of each antioxidant (KAO) is defined as the ratio of the
concentration of the antioxidant in the IL-rich (top) phase to that in the PVP-rich (bottom)
phase, as described by Equation 6,
IL-rich
phase
PVP-rich
phase
Figure 2.3-
Antioxidant
extraction with an
ABS composed of
IL + PVP + H2O.
48
𝐾𝐴𝑂 =[𝐴𝑂]𝐼𝐿
[𝐴𝑂]𝑃𝑉𝑃
where [AO]IL and [AO]PVP are, respectively, the concentrations of antioxidant in the IL-rich
and in the PVP-rich phases.
The extraction efficiencies of the antioxidants (EEAO%) are defined as the percentage
ratio between the amount of antioxidant in the IL-rich aqueous phase and the total amount
in the mixture, as defined in Equation 7,
𝐸𝐸𝐴𝑂% = [𝐴𝑂]𝐼𝐿 ∗ 𝑤𝐼𝐿
([𝐴𝑂]𝐼𝐿 ∗ 𝑤𝐼𝐿 + [𝐴𝑂]𝑃𝑉𝑃 ∗ 𝑤𝑃𝑉𝑃 )∗ 100
where [AO]IL and [AO]PVP are the amount of antioxidant in the IL-rich and in the PVP-rich
phases, and wIL and wPVP are the weight of each phase, respectively.
2.3. Results and discussion
2.3.1. Phase diagrams and tie-lines
In this section the results of the study of the ability of PVP with different molecular
weights to form ABS with several IL families as well as the effect of the IL nature are
described. The phase diagrams of each IL studied with each of the three different PVPs were
determined at (25 ± 1) °C and atmospheric pressure. For some of these combinations no
phase diagrams were obtained due to either complete miscibility or precipitation. The
different IL + PVP combinations used in the determination of a phase diagram are
summarized in Table 2.2, along with the information on the respective
successful/unsuccessful results. As it may be appreciated, from the different IL families
studied, only the phosphonium-based ILs were able to form ABS when combined with PVP.
It must be emphasized that this work presents the first evidence that the phosphonium-based
ILs form ABS with PVP and a detailed presentation of the results obtained follows.
(6)
(7)
49
Table 2.2- Combinations of the ILs and PVPs of different molecular weights that are able
(√) or not able, because of completely miscibility (m) or precipitation (p) to form ABS.
PVP10 PVP29 PVP40
[P4444]Br √ √ √
[P4444]Cl m m √
[Pi(444)1][Tos] √ √ √
[P4441][CH3SO4] √ √ √
[P4442][Et2PO4] √ √ √
[Ch][DHPh] p p p
[Ch]Cl m m m
[Ch][CH3CO2] p p p
[Ch][DHCit] m m m
[Ch][Bic] p p p
[Ch][Bit] m m m
[Ch][Prop] m
[Ch][Lac] m
[C2mim][ CH3CO2] m
[C2mim][HSO4] m
[C2mim][PO4(CH3)2] m
[C4mim][CF3SO3] p p p
[C4mim][SCN] m
[C4mim][BF4] p
[C4mim][Tos] m
[C6mim][N(CN)2] m
[N1120][CH3CO2] m m
[N11[2(N110)]0]Cl m m
[N11[2(N110)]0][CH3CO2] m m
From the ILs studied, only the phosphonium-based ILs were able to form ABS. Most
of the studied ILs, [Ch]Cl, [Ch][DHCit], [Ch][Bit], [Ch][Prop], [Ch][Lac],
[C2mim][CH3CO2], [C2mim][HSO4], [C2mim][PO4(CH3)2], [C4mim][SCN], [C4mim][Tos],
50
[C6mim][N(CN)2], [N1120][CH3CO2], [N11[2(N110)]0]Cl and [N11[2(N110)]0][CH3CO2], seem to
be completely miscible with the PVPs and the others, the [Ch][DHPh], [Ch][CH3CO2],
[Ch][Bic], [C4mim][CF3SO3] and [C4mim][BF4], precipitate when adding the IL solution to
the PVP solution (using the concentrations previously described).
As Table 2.2 evidences, a different behavior is observed for phosphonium-based ILs.
From the studied phosphonium-based ILs only the [P4444]Cl was unable to form ABS with
the lower molecular weight PVPs, and the two-phase splitting being observed only when it
is combined with PVP40. The phase diagrams obtained for every IL with each of the PVPs
(40,000 g.mol-1, 29,000 g.mol-1 and 10,000 g.mol-1) are depicted in Figures 2.4 to 2.6. All
the presented diagrams have the vertical axis to represent the PVP concentration and the
horizontal axis for the IL concentration. For all the systems the top phase is the IL-enriched
one, and the PVP-rich is the denser bottom phase. The binodal curves provide the minimum
concentration values of the phase forming components that are required to observe a two-
phase separation. Every mixture with compositions above the coexistent binodal curve
undergoes liquid-liquid demixing being within the biphasic region. A binodal curve closer
to the origin axis results in a larger biphasic region, hence a higher ABS forming capability.
Figure 2.4- Phase diagrams for the ternary systems composed of IL + PVP10 + H2O at 25 °C in mol.kg-1
units (left) and in wt.% (right): ▲, [P4444]Br; ♦, [P4441][CH3SO4]; ■, [P4442][Et2PO4]; ▲, [Pi(444)1][Tos].
All data in this section are displayed in weight percentage (wt. %) and in molality units
(mol.kg-1, moles of solute per kg of solvent) for a better perception of the distinct potential
for phase formation avoiding thus inconsistencies that may result from the various molecular
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0 10 20 30 40 50
[PV
P]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
0
10
20
30
40
25 40 55 70 85 100
[PV
P]
/ w
t. %
[IL]/ wt. %
51
weight variations of the solutes. The experimental weight fraction data for all the systems
are available in Appendix B (Tables B.1. to B.5.).
In Figure 2.4 are condensed the phase diagrams of the all ILs studied with PVP10.
From the data shown, the relative ability of the IL to form a biphasic system follows the
sequence: [P4444]Br > [P4441][CH3SO4] > [Pi(444)1][Tos] > [P4442][Et2PO4]. As mentioned
before, [P4444]Cl did not form a biphasic system with the PVP10, unlike the other ILs studied.
Figure 2.5- Phase diagrams for the ternary systems composed of IL + PVP29 + H2O at 25 °C in mol.kg-1
units (left) and in wt.% (right): ▲, [P4444]Br; ♦, [P4441][CH3SO4]; ■, [P4442][Et2PO4]; ▲, [Pi(444)1][Tos].
In Figure 2.5 are represented the phase diagrams of the ILs studied with the PVP29.
[P4444]Cl did not form a biphasic system with the PVP29 either. From the phase diagrams,
the ability of ILs to form a biphasic system can be ranked according to: [P4444]Br >
[P4441][CH3SO4] ≈ [Pi(444)1][Tos] > [P4442][Et2PO4].
The phase diagrams that combine the studied ILs with the highest molecular weight
PVP studied, the PVP40 are presented in Figure 2.6. From the depicted curves, it may be
inferred that the ILs ability to form a biphasic system follows the sequence: [P4444]Br >
[P4441][CH3SO4] ≈ [Pi(444)1][Tos] ≈ [P4442][Et2PO4] > [P4444]Cl. When the phase diagrams
are expressed in weight percentages instead of molalities, minor differences are observed in
the sequence of the ability of ABS forming: [P4444]Br > [P4441][CH3SO4] ≈ [P4444]Cl >
[Pi(444)1][Tos] ≈ [P4442][Et2PO4]. The small differences are observed for those ILs whose
phase diagrams are in close proximity but they are attenuated with the increase in the
5
15
25
35
25 35 45 55 65 75
[PV
P]
/ w
t. %
[IL]/ wt. %
0.001
0.006
0.011
0.016
0.021
1.0 3.0 5.0 7.0
[PV
P]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
52
molecular weight of PVP. Those small discrepancies may arise from intrinsic differences in
the interactions involved due to the polymer molecular weight.
Figure 2.6- Phase diagrams for the ternary systems composed of IL + PVP40 + H2O at 25 °C in mol.kg-1
units (left) and in wt.% (right): ▲, [P4444]Br; ●, [P4444]Cl; ♦, [P4441][CH3SO4]; ■, [P4442][Et2PO4]; ▲,
[Pi(444)1][Tos].
In spite of small discrepancies the trends are similar for the different molecular weight
polymers: [P4444]Br has the highest ability for two-phase promoting, and [P4444]Cl the lowest,
regardless of the polymer’s molecular weight. Since these ILs share the same cation, [P4444]+,
this seems to indicate that the anion that composes the IL has a marked influence in the
formation of ABS. The small differences in ABS forming among [P4441][CH3SO4],
[Pi(444)1][Tos] and [P4442][Et2PO4], decrease when the molecular weight of the PVP increases.
When comparing these results with the ones obtained in another study where the ability
of the same phosphonium-based ILs to form ABS with dextrans was determined [147], it
becomes evident that [P4444]Cl is a weaker ABS forming agent than the other ILs studied. In
that study [147], the phosphonium-based ILs were combined with three dextrans of
molecular weights 6 kDa, 40 kDa and 100 kDa, and [P4444]Cl was not able to form ABS with
dextran.
[P4442][Et2PO4] was also unable to form ABS with dextran [147], but could induce
phase separation with all the PVPs studied here. Gathering the data from that study and the
current one, [P4444]Cl appears to be the weakest ABS promoter, followed by [P4442][Et2PO4],
when combined with hydrophilic polymers. However, at the other end of the ranking order,
0.001
0.003
0.005
0.007
0.009
0.011
0.013
1 2 3 4 5 6 7 8 9
[PV
P]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
0
5
10
15
20
25
30
35
25 30 35 40 45 50 55 60 65 70 75[P
VP
] /
wt.
%[IL]/ wt.%
53
the similarities vanish: [P4444]Br was clearly the strongest ABS promoter when combined
with PVP, but just average when combined with either of the different molecular weight
dextrans. In fact, for the rest of the ILs a different order for the ability to promote ABS was
found in that study [147]: [Pi(444)1][Tos] > [P4444]Br > [P4441][CH3SO4], although the phase
diagrams of these systems are in close proximity.
When analyzing all data from a broader perspective, it may be inferred that the ability
to induce phase separation in ternary systems composed of IL + polymer + H2O, seems to
be higher for PVP than for dextran [147]. Both are neutral homopolymers, PVP a polar
water-soluble polymeric cyclic amide that presents high resistance to hydrolysis and dextran
a polysaccharide with a high number of hydroxyl groups, which have a great affinity for
water. PVP has a slightly more hydrophobic character than dextran, which may be
responsible for the observed behavior.
To better visualize the effect of the molecular weight of the polymer on ABS forming,
the phase diagrams of each IL with the three PVPs of the different molecular weights are
shown in Figures 2.7-2.10. [P4444]Cl is not represented, because it only forms ABS with the
largest PVP studied (40,000 g.mol-1).
The trends observed for all the ILs are similar: when the molecular weight of the PVP
increases, the ability to form ABS also increases. So, when the solubility of the polymer in
water increases there is a reduction on its affinity for water and it is easier to create an ABS,
being PVP40 the most efficient in ABS formation when combined with phosphonium-based
ILs.
54
Figure 2.7- Phase diagrams for ternary systems composed of [P4444]Br + PVP + H2O at 25 °C in mol.kg-1
units (left) and in wt.% (right): ♦, PVP10; ■, PVP29; ▲, PVP40.
Figure 2.8- Phase diagrams for ternary systems composed of [P4441][CH3SO4] + PVP + H2O at 25 °C in
mol.kg-1 units (left) and in wt.% (right): ♦, PVP10; ■, PVP29; ▲, PVP40.
0.00
0.01
0.02
0.03
0.04
0.05
0.06
1 2 3 4 5
[PV
P]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
5
10
15
20
25
30
35
40
25 30 35 40 45 50 55 60
[PV
P]
/ w
t.%
[IL]/ wt.%
0.001
0.003
0.005
0.007
0.009
0.011
0.013
0.015
0.017
3 4 5 6 7 8 9 10 11 12 13 14
[PV
P]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
2
7
12
17
22
53 58 63 68 73 78 83
[PV
P]
/ w
t.%
[IL]/ wt.%
55
Figure 2.9- Phase diagrams for ternary systems composed of [P4442][Et2PO4] + PVP + H2O at 25 °C in
mol.kg-1 units (left) and in wt.% (right): ♦, PVP10; ■, PVP29; ▲, PVP40.
Figure 2.10- Phase diagrams for ternary systems composed of [Pi(444)1][Tos] + PVP + H2O at 25 °C in
mol.kg-1 units (left) and in wt.% (right): ♦, PVP10; ■, PVP29; ▲, PVP40.
The experimental binodal curves were fitted to the empirical correlation described by
Merchuk et al. [144] (Equation 1). The regression parameters, the coefficients A, B and C,
and corresponding standard deviations and correlation coefficients (σ and R2, respectively)
were estimated by least squares regression for each ternary system and are reported in Table
2.3. Overall, satisfactory correlation coefficients were obtained and the corresponding
0.002
0.004
0.006
0.008
0.010
0.012
3 8 13 18 23 28 33 38 43 48
[PV
P]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
2
4
6
8
10
12
14
16
18
20
60 65 70 75 80 85 90 95
[PV
P]
/ w
t.%
[IL]/ wt.%
0.000
0.002
0.004
0.006
0.008
3.5 5.5 7.5 9.5 11.5 13.5
[PV
P]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
2
4
6
8
10
12
14
16
18
60 65 70 75 80 85
[PV
P]
/ w
t.%
[IL]/ wt.%
56
parameters are most useful to predict data in a given region of the phase diagram where no
experimental data are available.
Figure 2.11 and Figure 2.12 show that Equation 1 adequately fits to the experimental
data for the systems composed of [P4444]Br + PVP40 + H2O and [P4442][Et2PO4] + PVP40 +
H2O. The same behavior is observed for the other studied systems. It should be noted that
the TLs data were also used as experimental results when fitting Equation 1 to the binodal
curve, in order to enlarge the data amplitude and reliability. TLs were not determined by the
lever-arm rule as proposed by Merchuk et al. [144]; instead, they were experimentally
determined by quantitative analysis. Examples of the TLs obtained are shown in Figures
2.11 and 2.12 for the ternary systems composed of [P4444]Br and PVP40 and [P4442][Et2PO4]
and PVP40, respectively. In general, the TLs for each system are closely parallel to each
other, as Figure 2.11 and 2.12 show. All the remaining TLs are presented in Table 2.4.
57
Table 2.3- Correlation parameters used to describe the experimental binodal data by Equation 1 for the ABS
based on PVP and ILs.
IL + PVP + H2O A ± σ B ± σ 10-6(C ± σ) R2
[P4444]Br
PVP10 131.2 ± 8.0 -0.205 ± 0.013 4.88 ± 0.23 0.9984
PVP29 277.3 ± 20.8 -0.364 ± 0.016 3.89 ± 0.34 0.9985
PVP40 123.8 ± 1.5 -0.225 ± 2.894×10-3 7.35 ± 0.11 0.9994
[P4441][CH3SO4]
PVP10 208.4 ± 405.3 -0.082 ± 0.291 7.89 ± 1.44 0.9983
PVP29 310.8 ± 1188.0 -0.276 ± 0.599 4.12 ± 3.73 0.9959
PVP40 55.6 ± 29.9 -6.409×10-12 ± 0.094 6.48 ± 0.78 0.9911
[P4442][Et2PO4]
PVP10 93.6 ± 178.5 -8.714×10-11 ± 0.230 3.99 ± 0.52 0.9951
PVP29 150.9 ± 510.1 -6.705×10-10 ± 0.490 7.94 ± 2.09 0.9903
PVP40 153.5 ± 26.6 -9.200×10-12 ± 0.026 8.96 ± 0.23 0.9950
[Pi(444)1][Tos]
PVP10 399.2 ± 616.7 -5.110×10-14 ± 0.121 7.16 ± 1.14 0.9885
PVP29 56.9 ± 47.3 -1.618×10-11 ± 0.130 5.14 ± 0.75 0.9898
PVP40 59.9 ± 42.2 -8.379×10-12 ± 0.111 5.70 ± 0.68 0.9779
[P4444]Cl
PVP40 92.0 ± 98.1 -1.246×10-11 ± 0.157 8.48 ± 0.84 0.9829
58
Figure 2.11- Phase diagram for the ternary system composed of [P4444]Br + PVP40 + H2O: binodal curve
data (▲); TL 1 data (X); TL 2 data (●); adjusted binodal data through Equation 1 (―).
Figure 2.12- Phase diagram for the ternary system composed of [P4442][Et2PO4] + PVP40 + H2O: binodal
curve data (▲); TL 1 data (X); TL 2 data (●); adjusted binodal data through Equation 1 (―).
To evaluate the consistency of the experimentally obtained TLs, their deviation was
ascertained for one system. To that aim, three TLs combining [P4444]Br and PVP40 with the
composition 67 wt. % of IL + 13 wt. % of PVP + 20 wt. % of H2O were determined, and the
0
10
20
30
40
50
60
70
5 15 25 35 45 55 65 75 85
[PV
P]
/ w
t. %
[IL] / wt. %
0
5
10
15
20
25
30
35
55 60 65 70 75 80
[PV
P]
/ w
t. %
[IL] / wt. %
59
deviation results are represented in Table 2.4 for the correspondent TL. It was found that the
deviations are not significant. The greatest absolute deviations were found to be in the weight
fraction composition of the PVP-rich phase, 1.62 and 0.80 wt. % for the fraction of IL and
PVP, respectively. In the IL-rich phase the experimentally determined weight fractions are
more consistent, having absolute deviations of 0.08 and 0.48 wt. % for the PVP and the IL
fraction, respectively.
60
Table 2.4- Experimental data for TLs of the ABS IL + PVP + H2O.
IL + PVP +
H2O
Weight fraction composition / wt. %
[PVP]IL [IL]IL [PVP]M [IL]M [PVP]PVP [IL]PVP
[P4444]Br
PVP10 1.00 80.39 13.01 66.27 51.47 19.42
1.27 75.02 29.85 42.72 41.76 26.84
PVP29 3.08 79.41 13.06 67.06 40.52 21.05
4.00 71.99 30.03 39.97 42.06 22.89
PVP40 3.85 60.70 14.68 46.41 21.40 36.77
0.30±0.08 81.14±0.48 12.85 66.60 63.46±0.80 8.75±1.62
[P4441][CH3SO4]
PVP10 2.08 80.44 32.95 62.58 22.51 58.69
PVP29 1.94 74.97 15.92 60.86 22.34 53.44
PVP40 1.52 77.52 12.99 66.91 36.49 39.18
1.85 75.50 15.03 62.81 26.15 50.35
[P4442][Et2PO4]
PVP29 1.40 79.86 15.99 66.82 21.65 62.08
PVP40 1.30 79.49 13.00 66.76 20.64 60.93
1.71 77.80 13.07 65.96 17.61 62.14
[Pi(444)1][Tos]
PVP29 4.69 78.36 15.07 66.93 32.93 46.94
PVP40 6.66 74.32 13.01 66.91 25.76 53.42
1.31 82.81 13.11 69.69 41.15 38.44
[P4444]Cl
PVP40 0.69 78.48 11.08 65.86 20.71 57.23
0.05 79.76 13.01 66.91 24.82 53.90
61
2.3.2. Partition of antioxidants
As mentioned before, ABS are a promising tool for biomolecules extraction. So, after
the complete characterization of the novel IL-PVP ABS, they were further evaluated in what
concerns their ability to extract antioxidants, namely gallic acid (pKa = 3.94; 9.04; 11.17)
[14], syringic acid (pKa = 3.93; 9.55) [14], vanillin (pKa = 7.81) [14], and vanillic acid (pKa
= 4.16; 10.14) [14]. In order to minimize deviations that might arise from differences in the
ternary mixture compositions, the partition experiments of the studied antioxidants were
accomplished at a fixed mixture composition: 67 wt. % of IL + 13 wt. % of PVP + 20 wt. %
of antioxidant solution. The pH values of each phase (top and bottom) of the extracting
systems using the three molecular weight PVPs are presented in Table 2.5. The pH was
measured after the separation of the two phases and the presented values are the average of
the values measured for each system. Within the same ILs, the pH values were almost the
same for the four antioxidants and varied only for the different ILs.
Table 2.5- Average pH values of the bottom (pHPVP) and the top (pHIL) phase of the ABS used for the
extraction of the antioxidants.
Polymer IL pHPVP pHIL
PVP40
[P4444]Br 1.26 ± 0.03 1.27 ± 0.02
[P4441][CH3SO4] 2.36 ± 0.04 1.55 ± 0.02
[Pi(444)1][Tos] 2.05 ± 0.07 1.53 ± 0.01
[P4444]Cl 5.31 ± 0.16 3.50 ± 0.05
[P4442][Et2PO4] 5.10 ± 0.05 2.77 ± 0.11
PVP29 [P4444]Br 1.97 ± 0.12 2.58 ± 0.13
PVP10 [P4444]Br 1.74 ± 0.12 2.29 ± 0.11
The results obtained for the extraction of each antioxidant using the five phosphonium-
based ILs are summarized in Figure 2.13 (partition coefficients) and Figure 2.14 (extraction
efficiencies). The pH values measured for the two phases of the extracting systems are also
shown. The complete set of values of the K and EE% are presented in Appendix C.
62
Figure 2.13- Partition coefficients of gallic acid, vanillin, vanillic acid and syringic acid in a biphasic system
composed of 67 wt.% IL + 13 wt.% PVP40 + 20 wt.% antioxidant aqueous solution at 25 °C and average pH
values of the IL-rich phase (Top) and PVP-rich phase (Bottom).
1
2
3
4
5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
[P4444]Br [P4441][MeSO4] [Pi(444)1][TOS] [P4442][Et2PO4] [P4444]Cl
pHK
Gallic acid Syringic acid Vanilin Vanillic acid pH Top pH Bottom
[P4444]Br [P4441][CH3SO4] [Pi(444)1][Tos] [P4442][Et2PO4] [P4444]Cl
63
Figure 2.14- Extraction efficiency of gallic acid, vanillin, vanillic acid and syringic acid in a biphasic system
composed of 67 wt. % of IL + 13 wt.% of PVP40 + 20 wt. % of antioxidant aqueous solution at 25 °C and
pH values of the IL-rich phase (Top) and PVP-rich phase (Bottom).
As it may be appreciated from the data shown, the values of K and EE%, when using
[P4444]Br, increase in the sequence: gallic acid < syringic acid < vanillin < vanillic acid. Both
ABS phases have an acidic pH of ca. 1.26 ± 0.03 at the bottom phase (PVP-rich phase) and
1.27 ± 0.02 at the top phase (IL-rich phase), independently of the molecule being extracted.
At this pH value the antioxidants are mostly in their neutral form; the graphs showing the
concentration of the antioxidants at different pH values are presented in Appendix C.
Regarding the log (Kow) values of the antioxidants it is evident that for this IL, the
EE% and K increases with the increase of hydrophobicity, since the log (Kow) values are
0.70, 1.04, 121, and 1.43 for the gallic acid, syringic acid, vanillin and vanillic acid,
respectively [14].
A resembling speciation is to be found in the extractions using [P4441][CH3SO4] and
[Pi(444)1][Tos], since the pH values of the aqueous phases involved are similar, the pH of
[P4441][CH3SO4] and [Pi(444)1][Tos] being 2.36 ± 0.04 and 2.05 ± 0.07 for the bottom phase
and 1.55 ± 0.02 and 1.53 ± 0.01 for the top phase, respectively. Although pH values are
1
2
3
4
5
55
60
65
70
75
80
85
90
95
100
[P4444]Br [P4441][MeSO4] [Pi(444)1][TOS] [P4442][Et2PO4] [P4444]Cl
pHEE%
Gallic acid Syringic acid Vanillin Vanillic acid pH Top pH Bottom
[P4444]Br [P4441][CH3SO4] [Pi(444)1][Tos] [P4442][Et2PO4] [P4444]Cl
64
slightly higher and there is a small divergence between the pH values of the two ABS phases,
these small disparities should not lead to qualitative changes in the major forms of the
extracted molecule, the neutral form persisting as the most abundant in every case. In spite
of this, the values of K and EE% for [P4441][CH3SO4] and [Pi(444)1][Tos] follow a reverse
order from the one observed for [P4444]Br and decrease in the sequence: gallic acid > syringic
acid > vanillin > vanillic acid. This inversion cannot be imparted to charge differences or
electrostatic effects, since the speciation of the extracted molecule is identical for the three
ILs, so it must originate from intrinsic affinity differences among the phase components (IL
anion and cation and polymer) and the extracted molecule. In this case, the more hydrophilic
antioxidants have a higher affinity to the IL-rich phase than the hydrophobic ones.
[P4444]Br appears as the most efficient agent for extracting the antioxidants, apart from
gallic acid; this is not unexpected since this IL is also the strongest ABS promoter.
The interpretation of the results obtained with [P4442][Et2PO4] and the [P4444]Cl is far
more intricate since several factors may influence the process. In fact, not only pH values
are higher than in the previous cases, but the differences between the two aqueous phases
are no longer negligible, which means that the antioxidant speciation is different in each
phase of the ABS. For extractions with [P4442][Et2PO4] and [P4444]Cl, the pH values of the
bottom phase are 5.10 ± 0.05 and 5.31 ± 0.16, respectively; at these pH values only the
vanillin is neutral, all the other three antioxidants are negatively charged. For the top phases
the corresponding pH values are 2.77 ± 0.11 and 3.50 ± 0.05, respectively, and all the
antioxidants are neutral. So, charge effects may superimpose to intrinsic affinities, and the
overall effects lead to the following sequence: syringic acid < gallic acid < vanillin < vanillic
acid, where the values when using [P4442][Et2PO4] are slightly higher than when using
[P4444]Cl. Here the antioxidants do not follow the hydrophobicity sequence. Nevertheless,
regarding the values of gallic acid and syringic acid, that are very proximal taking into
account the associated standard deviations, it is not that evident if the values are the same or
which one is higher.
On the whole, the phosphonium-based ILs combined with PVP presented K values
greater than unity for the extraction of the studied antioxidants, which shows that these
molecules have an enhanced affinity for the IL than for the polymer. The extraction
efficiencies varied from nearly 70 % to 92 %, with [P4444]Br emerging as a valuable choice
particularly for extracting vanillic acid. If we consider the effect of the IL anion
65
independently, we infer that Br- seems to have a stronger effect in “attracting” vanillin to the
IL phase than Cl-. This result is opposite to that observed in the extraction of the same
antioxidant, vanillin, using imidazolium-based ILs combined with K3PO4 [124]. Although
these may seem contradictory results, in fact it is not so, because the difference in the pH in
both studies, basic and higher than vanillin acidity constant when the phosphate salt is used
and moderately acidic and lower than that acidity constant in this work, may certainly alter
the relative affinities of vanillin for both aqueous phases. A more complete study on the
effects at stake, such as pH and individual IL cation/anion effects, needs to be performed in
the future so as to shed some light in some apparent contradictions observed along this study.
In the following Figures 2.15 and 2.16 are represented the K and EE% values for the
extractions of the antioxidants gallic acid, vanillic acid, vanillin and syringic acid performed
by [P4444]Br combined with the three PVPs studied.
[P4444]Br was chosen for the extractions since this IL revealed the highest ability to
form ABS when combined with every PVP studied regardless of its molecular weight.
Moreover, for this IL it was possible to perform the antioxidants extractions using an ABS
composition that was analogous to the one used for the PVP40, which allows a
straightforward comparison among the results.
66
Figure 2.15- Partition coefficient of gallic acid, vanillin, vanillic acid and syringic acid and the pH values of
the IL-rich phase (pH Top) and the PVP-rich phase (pH Bottom) in a biphasic system composed of 67 wt.%
[P4444]Br + 13 wt.% PVP + 20 wt.% antioxidant aqueous solution at 25 °C.
In Figure 2.15 it is shown that the partition coefficient of the antioxidants with the
[P4444]Br and the different PVPs follows the sequence: PVP10 > PVP29 > PVP40 for gallic
acid and vanillin. For these two antioxidants the decrease of the molecular weight of PVP is
favorable for their partitioning into the IL-rich phase. Nevertheless, for syringic acid and
vanillic acid the opposite is shown. The K values increase with the increase of the molecular
weight of PVP and according to the rank: PVP40 > PVP29 > PVP10. For both the PVP10
and PVP29 the highest K was obtained for the extraction of vanillin (3.18 and 3.11,
respectively), but when using PVP40 the highest value was obtained in the extraction of
vanillic acid (4.24).
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
gallic acid syringic acid
pHK
PVP40 PVP29 PVP10 pH Top pH Bottom
Gallic acid Syringic acid Vanillin Vanillic acid
67
Figure 2.16- Extraction efficiency of gallic acid, vanillin, vanillic acid and syringic acid and the pH values of
the IL-rich phase (pH Top) and the PVP-rich phase (pH Bottom) in a biphasic system composed of 67 wt.%
[P4444]Br + 13 wt.% PVP + 20 wt.% antioxidant aqueous solution at 25 °C.
When analyzing EE% values (Figure 2.16) the same sequences can be observed:
PVP10 > PVP29 > PVP40 for gallic acid and vanillin and PVP40 > PVP29 > PVP10 for
syringic acid and vanillic acid. As observed before for partition coefficients, also the highest
values were obtained for EE% with the PVP10 and PVP29 in the extraction of vanillin
(91.46% and 89.87%, respectively) and for PVP40 in the extraction of vanillic acid
(92.00%). Actually, the molecular structure of both antioxidants are similar and small
differences between them may have a great influence in the extraction mechanism. The pH
values for the phases are almost the same for the systems with the same IL and the different
antioxidants, varying only for the different PVPs, that are 1.26 ± 0.03, 1.97 ± 0.12 and 1.74
± 0.12 for the PVP-rich phases of PVP40, PVP29 and PVP10, respectively. For the IL-rich
phases the pH values are 1.27 ± 0.02, 2.58 ± 0.13 and 2.29 ±0.11 for the PVP40, PVP29 and
PVP10, respectively. According to the pKa values, at these pHs the antioxidants are all non-
charged.
0.5
1.0
1.5
2.0
2.5
3.0
70
75
80
85
90
95
gallic acid syringic acid
pHEE%
PVP40 PVP29 PVP10 pH Top pH Bottom
Gallic acid Syringic acid Vanillin Vanillic acid
68
The EE% of the antioxidants in this study, where the highest obtained value was 92
%, are not as high as obtained with some other studies previously reported with systems
composed of ILs, where it were obtained EE% up to 99 % [123,132]. Nevertheless the
obtained EE% in this study is a promising result, since different compositions may lead to
better results. In this work only a fixed ABS composition was investigated for all the
systems. The optimization of the extraction of the antioxidants with the ABS composed of
ILs and PVP should be performed next, testing other compositions to perform the extraction
in order to obtain better extraction results. In fact, the ABS studied in this work have several
advantages when compared to ABS used before to extract antioxidants. A major advantage
is the avoidance of inorganic salts that may be harmful to the environment or even to the
extracted biomolecules [101]. The mentioned EE% of up to 99% that were obtained in the
extraction of antioxidants (gallic acid, vanillic acid and syringic acid) used systems
composed of the inorganic salt Na2SO4 [123,132]. However, the results here obtained are
much better than the ones obtained in the extraction of antioxidants (syringic acid and
vanillin) using ABS constituted of two polymers [104]. In that work, using ABS without
ILs, values of EE% were never higher than 80%, a value significantly poorer than those
obtained here using IL-based ABS.
69
2.4. Conclusions
This study reports, for the first time, the ability of three different molecular weight
PVPs, 10,000 g.mol-1, 29,000 g.mol-1 and 40,000 g.mol-1, to form ABS combined with
phosphonium-based ILs. A few previous studies report the formation of ABS composed of
phosphonium-based ILs, but those ILs were combined with stronger ABS agents, as are
inorganic salts, K3PO4 [145], Al2(SO4)3 [148] and K3C6H5O7 [148] and also with a
biopolymer, dextran [147].
The phase diagrams and respective TLs were determined for novel ABS with five
phosphonium-based ILs combined with three molecular weight PVPs. The results
established [P4444]Br as the strongest ABS promoter and [P4444]Cl the weakest. The nature
of both the cation and the anion of the IL seem to influence the ability to form ABS.
The molecular weight of the polymer also influences the two phase forming; the larger
the molecular weight of the polymer the stronger the ABS forming ability. This behavior is
similar to what was observed with dextran [147], also a neutral hydrophilic polymer.
[P4444]Br was shown to have the best extraction efficiency for all the antioxidants
studied, syringic acid, vanillin and vanillic acid, except for gallic acid that was more
extensively extracted using [P4441][CH3SO4]. These results seem to indicate some correlation
between the ABS promoting ability and extracting ability, but other influences are also
playing a role.
The EE% obtained in this study (up to 92%) are higher than those obtained with an
ABS composed of two polymers where the highest value obtained was 80%. [104]. But in
the literature there are also studies that reported higher EE% (up to 99%) [123,132] than
those of this study, but those systems used the inorganic salt Na2SO4 for the formation of the
ABS, which may be inconvenient.
The systems here studied, that combine polymers with phosphonium-based ionic
liquids seem to be a valuable alternative to other ABS since they combine the mild charge
of ILs with the neutrality of polymers, achieving the phase’s polarity difference by means of
polymer weight and IL nature.
70
71
3. IL + PVA + H2O ABS
72
73
3.1. Poly(vinyl alcohol) (PVA)
Poly(vinyl alcohol) (PVA) is a polymer that is synthetically produced by removing
the acetate groups of the poly(vinyl acetate) (PVAc) [149] which is then hydrolyzed to get
PVA. The structure of PVA is given in Figure 3.1; it has a hydroxyl group in its monomeric
structure. The crystallizability and solubility of PVA is affected by the extent of hydrolysis
and content of acetate groups [150]. It is soluble in solvents, such as water, dimethyl
sulfoxide and ethylene glycol [150]; the solubility of PVA in water depends on a variety of
parameters, such as the degree of polymerization, hydrolysis and temperature of the solution.
Any change in these three factors affects the degree and the character of hydrogen bonding
in the aqueous solutions, and hence the solubility of PVA. The increase of the degrees of
hydrolysis of PVA decreases the solubility in water [150]. Besides solubility, also viscosity
and surface tension of PVA depend on temperature, concentration, hydrolysis extent and
molecular weight of the material [151].
Figure 3.1- Chemical structure of PVA.
PVA can form hydrogels, for example by techniques such as ‘‘salting-out’’ polymer
gelation [149]. For the formation of the hydrogels the polymer must be cross-linked and then
it becomes less soluble [151]. PVA gels swell in water and biological fluids and are well
accepted by the human body and that’s why the PVA can be used, for example, in contact
lenses, drug delivery systems and for the substitution of soft tissue [151]. It is not only used
in medical devices, but due to its properties, namely high solubility in water, chemical
resistance, biodegradability, non-toxicity and the ease of usage, PVA is also used in a wide
range of applications, for example in the textile, pharmaceutical and food packaging
industries [149,151].
PVA was already used in the formation of ABS, for example combined with the
polysaccharide dextran [141]; it was shown that adding an aqueous solution of an inorganic
74
salt, such as KSCN and K2SO4, changes the ability of the system composed of dextran and
PVA to form ABS.
It was also reported that PVA combines with 1-butyl-3-methylimidazolium
tetrafluoroborate, [C4mim][BF4], to form an hydrogel [152]. In the presence of the IL the
PVA gel shrinks, because water is released from the gel; this process is reversible, since
when water is added, the gel swells again [152].
75
3.2. Experimental section
3.2.1. Chemicals
Poly(vinyl alcohol) (PVA) of four different molecular weights, 9,000-10,000 g.mol-1
(PVA9), 13,000-23,000 g.mol-1 (PVA13), 30,000-70,000 g.mol-1 (PVA30) and 85,000-
124,000 g.mol-1 (PVA85) were used and purchased from Sigma-Aldrich. The structure of
PVA is represented in Figure 3.1.
The antioxidants and ILs investigated were those presented and described in Section
2.2.1.
Water was distilled twice, passed by a reverse osmosis system and additional treated
with a Milli-Q plus 185 water purification apparatus.
3.2.2. Experimental procedure
3.2.2.1 Phase diagrams and tie-lines
For the determination of the phase diagrams the cloud point titration method at (25 ±
1) °C and atmospheric pressure was used, according to the procedure described in section
2.2.2.1. Aqueous solutions of PVA9 (33 wt. %), PVA13 (28 wt. %), PVP30 (22 wt. %) and
PVA85 (13 wt. %) and aqueous solutions of ILs at different concentrations (80-100 wt. %)
were initially prepared. Due to the precipitation of some ILs when added to the solutions of
PVA at these concentration levels, less concentrated PVA aqueous solutions were then
prepared. The phase diagrams of PVA9, PVA13, PVA30 and PVA85 were then obtained
using solutions around 5 wt. %. To determine the phase diagrams, the IL solution was drop-
wise added to the PVA solution until the solution becomes cloudy and then water was drop-
wise added until the system becomes clear. This process was repeated under constant
stirring. The tie-lines (TL) were prepared using the method described by Merchuk et al. [144]
and that was mentioned in section 2.2.2.1.
76
The pH values of both the IL-rich and the PVA-rich phases were measured at (25 ± 1)
°C using a Mettler Toledo S47 SevenMultiTM dual meter pH/conductivity equipment with
an uncertainly of ± 0.02 pH units.
3.2.2.2 Partition of antioxidants
For the extraction of the antioxidants using ABS composed of PVA and the ILs,
aqueous solutions of gallic acid (1 g.dm-3), vanillin (1 g.dm-3), vanillic acid (0.5 g.dm-3) and
syringic acid (0.5 g.dm-3) were prepared.
The extraction of the antioxidants was studied within a fixed
composition in the biphasic region of each system: 58 wt. % of IL + 3
wt. % of PVA + 39 wt. % of antioxidant solution (Figure 3.2). For the
system composed of PVA85 and [C4mim][BF4] precipitation occurred
upon IL and polymer mixing, and a different composition was used: 43
wt. % of IL + 2 wt. % of PVA + 55 wt. % of antioxidant solution. After
mixing the required amounts of solutions, they were vigorously
agitated until dissolution. For the complete separation of the two
phases, each mixture was left at rest at least for 12 hours at 25 °C. After
that period of time, the two phases were carefully separated and
weighted, the pH of each phase was measured and the concentration of antioxidant at each
phase was determined. At least two independent samples were prepared and analyzed for
each system.
The quantification of the antioxidant in each phase was performed by UV-spectroscopy,
carried out with a SHIMADZU UV-1800 spectrophotometer, at a wavelength of 262 nm for
gallic acid, 274 nm for syringic acid, 280 for vanillin and 292 nm for vanillic acid; a blank control
sample was always used. The calibration curves for each antioxidant are presented in Appendix
A.
The partition coefficients of each antioxidant (KAO) and extraction efficiencies of the
antioxidants (EEAO%) were calculated according to Equations 6 and 7 described in section
2.2.2.3.
PVA-rich phase
IL-rich
phase
Figure 3.2-
Antioxidant
extraction with an
ABS composed of
IL + PVA + H2O.
77
3.3. Results and discussion
3.3.1. Phase diagrams and tie-lines
In this section the ability of ILs to form ABS with PVA of four different molecular
weights, PVA9, PVA13, PVA30 and PVA85, was investigated. In Table 3.1 the systems that
have been studied are reported as well as the corresponding results, that is, whether or not
they were successful.
78
Table 3.1- Combinations of the ILs and PVAs of different molecular weights that are able (√) or not able,
because of completely miscibility (m) or precipitation (p), to form ABS.
PVA9 PVA13 PVA30 PVA85
[C2mim][CH3CO2] m m
[C2mim][HSO4] m m
[C2mim][PO4(CH3)2] m m
[C4mim][CF3SO3] √ √ √ √
[C4mim][SCN] m m
[C4mim][BF4] √ √ √ √
[C4mim][Tos] m m
[C4mim][PO4(CH3)2] m m
[C4mim][HSO4] m m
[C6mim][N(CN)2] m m
[P4444]Br m m
[P4444]Cl m
[Pi(444)1][Tos] m m
[P4441][CH3SO4] m m
[P4442][Et2PO4] m m
[Ch][DHPh] p p p p
[Ch]Cl p p p p
[Ch][ CH3CO2] p p p p
[Ch][Bit] p p
[Ch][DHCit] p p
[Ch][Prop] m m
[Ch][Lac] p p
[Ch][Bic] p p p p
[N11[2(N110)]0]Cl p p
[N11[2(N110)]0][CH3CO2] m m
[N1120][CH3CO2] m m
79
As it may be appreciated, only two of the studied ILs were able to form ABS with
PVA. For the following ILs, [C2mim][CH3CO2], [C2mim][HSO4], [C2mim][PO4(CH3)2],
[C4mim][SCN], [C4mim][Tos], [C4mim][PO4(CH3)2], [C4mim][HSO4],[C6mim][N(CN)2],
[P4444]Br, [P4444]Cl, [Pi(444)1][Tos], [P4441][CH3SO4], [P4442][Et2PO4], [Ch][Prop],
[N11[2(N110)]0][CH3CO2] and [N1120][CH3CO2] cloudiness could not be achieved.
At first, the systems combining PVA with [C4mim][CF3SO3], [C4mim][BF4], [Ch]Cl,
[Ch][DHPh], [Ch][CH3CO2], [Ch][DHCit] and [Ch][Bic] seemed to form ABS, because
upon the addition of the IL solution to the PVA solution, the system became cloudy and the
addition of water made the solution clear again - the usual behavior of
a typical ABS. Nevertheless, when trying to make a solution within
the biphasic region of [Ch][CH3CO2] it was noticed that cloudiness
was not due to the formation of a biphasic system but to the
precipitation that occurs when adding the IL solution to the PVA
solution (Figure 3.3). Consequently, the same procedure was followed
to investigate the behavior of [Ch][Bic], [Ch][DHPh] and
[C4mim][BF4] and precipitation was also observed.
The eventual formation of a precipitate when using the cloud
point titration method for the ILs was thoroughly investigated. The
procedure was repeated, but after obtaining a few points of the
solubility curve, instead of performing the usual procedure, adding
water and stopping the addition of IL when the solution becomes cloudy, a few more drops
of IL solution were added to observe if a precipitate would form. And indeed it was observed
that precipitation occurs for many systems involving PVA and ILs, namely with
[Ch][DHPh], [Ch]Cl, [Ch][CH3CO2], [Ch][Bit], [Ch][DHCit], [Ch][Lac], [Ch][Bic] and
[N11[2(N110)]0]Cl.
In order to avoid the possibility of precipitate formation upon adding the IL solution,
the concentrations of the PVA aqueous solutions used were decreased, and experiments were
made to observe weather the precipitation persisted or not. PVA solutions at 5 wt. % were
then prepared and used in the cloud point titrations. For [C4mim][CF3SO3] and
[C4mim][BF4] it was possible to observe ABS formation when combined with PVA of either
molecular weight; the tie-lines could also be prepared, and no precipitation was observed. In
Figure 3.4 and Figure 3.5 the phase diagrams of the [C4mim][CF3SO3] and [C4mim][BF4]
Figure 3.3-
Precipitation of the
system composed of
IL + PVA + H2O.
80
with the PVAs of the four molecular weights are depicted. Due to the wide range of the
molecular weight values for these PVAs, the average of the molecular weight was
determined for each PVA to create the phase diagrams in mol.kg-1 units.
All the following phase diagrams have the vertical axis to represent the PVA
concentration and the horizontal axis for the IL concentration. For all the systems the top
phase is the PVA-enriched one, and the denser bottom phase is the IL-rich phase. All data
in this section are displayed in weight percentage (wt. %) and in molality units (mol.kg-1,
moles of solute per kg of solvent). The experimental weight fraction data for all the systems
are available in Appendix B (Tables B.6 and B.7).
Figure 3.4- Phase diagrams for the ternary systems composed of [C4mim][CF3SO3] + PVA + H2O at 25 °C
in mol.kg-1 units (left) and in wt.% (right): ■, PVA85; ▲, PVA30; x, PVA13; ♦, PVA9.
0.000
0.001
0.002
0.003
0.004
0.005
0.006
2 3 4 5 6 7 8
[PV
A]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
0
1
2
3
4
40 45 50 55 60
[PV
A]
/ w
t. %
[IL]/ wt. %
0.0000
0.0004
2.5 3.0 3.5
81
Figure 3.5- Phase diagrams for the ternary systems composed of [C4mim][BF4] + PVA + H2O at 25 °C in
mol.kg-1 units (left) and in wt.% (right): ■, PVA85; ▲, PVA30; x, PVA13; ♦, PVA9.
As it can be seen in Figures 3.4 and 3.5, the ability to form ABS increases, for both
ILs, [C4mim][CF3SO3] and [C4mim][BF4], with the increase of the molecular weight of
PVA: PVA85 > PVA30 > PVA13 > PVA9. This behavior evidences the decrease of PVA’s
affinity for water when the molecular weight increases and it is identical to the one
previously observed for ABS composed of PVP and phosphonium-based ILs.
The comparison of the phase forming ability of the two ILs with the four PVAs studied
are shown in Figure 3.6 to Figure 3.9. It can be seen that for all the PVAs, the [C4mim][BF4],
that has a more hydrophobic anion (log (Kow) of [BF4]- = 0.22 and log (Kow) of [CF3SO3]
- =
-0.49 [14]) has a greater ability to form a biphasic system when compared to
[C4mim][CF3SO3]. The same behavior was previously shown with ABS composed of these
ILs and the amino acids L-proline and L-lysine [153] and for those based on maltodextrin
[147].
0.000
0.001
0.002
0.003
0.004
0.005
0.006
2 3 4 5 6
[PV
A]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
0
1
2
3
4
5
6
32 37 42 47 52 57
[PV
A]
/ w
t. %
[IL]/ wt. %
0.0000
0.0003
2.2 2.5 2.8
0.5
1.0
1.5
34 35 36 37
82
Figure 3.6- Phase diagrams for the ternary systems composed of IL + PVA9 + H2O at 25 °C in mol.kg-1 units
(left) and in wt.% (right): ■, [C4mim][BF4]; ♦, [C4mim][CF3SO3].
Figure 3.7- Phase diagrams for the ternary systems composed of IL + PVA13 + H2O at 25 °C in mol.kg-1
units (left) and in wt.% (right): ■, [C4mim][BF4]; ♦, [C4mim][CF3SO3].
0.000
0.001
0.002
0.003
0.004
0.005
0.006
3.2 3.5 3.8 4.1 4.4 4.7 5.0
[PV
A]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
0.5
1.5
2.5
3.5
4.5
5.5
42 45 48 51 54 57 60
[PV
A]
/ w
t. %
[IL]/ wt. %
0.0004
0.0008
0.0012
0.0016
0.0020
2.3 2.6 2.9 3.2 3.5 3.8
[PV
A]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
0.5
1.0
1.5
2.0
2.5
3.0
3.5
35 38 41 44 47 50 53
[PV
A]
/ w
t. %
[IL]/ wt. %
83
Figure 3.8- Phase diagrams for the ternary systems composed of IL + PVA30 + H2O at 25 °C in mol.kg-1
units (left) and in wt.% (right): ■, [C4mim][BF4]; ♦, [C4mim][CF3SO3].
Figure 3.9- Phase diagrams for the ternary systems composed of IL + PVA85 + H2O at 25 °C in mol.kg-1
units (left) and in wt.% (right): ■, [C4mim][BF4]; ♦, [C4mim][CF3SO3].
The experimental binodal curves were fitted to the empirical correlation described by
Merchuk et al. [144] that is represented in Equation 1 in section 2.2.2.1. The regression
parameters, the coefficients A, B and C, and corresponding standard deviations and
correlation coefficients (σ and R2, respectively) were estimated by least squares regression
for each ternary system and are shown in Table 3.2. In general, satisfactory correlation
coefficients were obtained; the corresponding parameters are indeed valuable to predict data
in the regions of the phase diagram where no experimental data are available.
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
2.0 2.4 2.8 3.2 3.6
[PV
A]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
0.5
1.0
1.5
2.0
2.5
3.0
32 35 38 41 44 47 50
[PV
A]
/ w
t. %
[IL]/ wt. %
0.00004
0.00008
0.00012
0.00016
0.00020
2.2 2.6 3.0 3.4
[PV
A]
/ m
ol.
kg
-1
[IL]/ mol.kg-1
0.4
0.8
1.2
1.6
2.0
32 36 40 44 48
[PV
A]
/ w
t. %
[IL]/ wt. %
84
Two examples of the adequacy of the correlation of Equation 1 with experimental data
for the systems composed of [C4mim][BF4] + PVA30 + H2O and [C4mim][CF3SO3] +
PVA13 + H2O are shown Figure 3.10 and Figure 3.11. The same behavior is observed for
the other studied systems.
The TLs were determined by the lever-arm rule as proposed by Merchuk et al. [144];
examples of the TLs obtained for the systems composed of [C4mim][BF4] + PVA30 + H2O
and [C4mim][CF3SO3] + PVA13 + H2O are shown in the Figures 3.10 and 3.11, respectively.
In general, the TLs for each system are closely parallel to each other. All the remaining TLs
data are presented in Table 3.3.
Table 3.2- Correlation parameters used to describe the experimental binodal data by Equation 1 for the ABS
based on IL and PVA.
IL + PVA + H2O 102(A ± σ) B ± σ 10-5(C ± σ) R2
[C4mim][BF4]
PVA85 73.6 ± 26.7 -0.340 ± 7.310 14.91 ± 16.26 0.9953
PVA30 918.3 ± 35840.0 -1.305 ± 7.916 7.77 ± 18.14 0.9647
PVA13 103.2 ± 4260.0 -0.715 ± 8.106 8.22 ± 15.83 0.9842
PVA9 695.0 ± 17080.0 -0.814 ± 4.383 5.09 ± 5.28 0.9793
[C4mim][CF3SO3]
PVA85 7.8 ± 1.4 -6.367×10-11 ± 0.022 6.30 ± 0.16 0.9968
PVA30 3.0 ± 13.0 -0.567 ± 0.787 1.77 ± 1.01 0.9970
PVA13 4.1 ± 69.5 -0.672 ± 2.931 0.74 ± 3.05 0.9785
PVA9 38.7 ± 12.5 -6.596×10-13 ± 0.021 3.95 ± 0.16 0.9806
85
Figure 3.10- Phase diagram for the ternary system composed of [C4mim][BF4] + PVA30 + H2O: binodal
curve data (▲); TL 1 data (X); TL 2 data (●); adjusted binodal data through Equation 1 (―).
Figure 3.11- Phase diagram for the ternary system composed of [C4mim][CF3SO3] + PVA13 + H2O: binodal
curve data (▲); TL 1 data (X); TL 2 data (●); adjusted binodal data through Equation 1 (―).
0
5
10
15
20
25
25 30 35 40 45
[PV
A]
/ w
t. %
[IL] / wt. %
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0
[PV
A]
/ w
t. %
[IL] / wt. %
86
Table 3.3- Experimental data for TLs of the ABS IL + PVA + H2O.
IL + PVA + H2O Weight fraction composition / wt. %
[PVA]IL [IL]IL [PVA]M [IL]M [PVA]PVP [IL]PVP
[C4mim][BF4]
PVA85 0.06 40.03 1.21 38.38 4.99 32.97
0.00 46.89 2.24 42.93 8.48 31.91
PVA30 0.09 41.30 1.21 40.68 26.88 26.41
0.15 40.18 1.37 39.54 26.11 26.51
PVA13 0.13 43.07 1.39 41.76 10.25 32.56
0.01 47.10 1.42 45.20 10.98 32.34
PVA9 0.96 47.81 2.01 47.04 9.34 41.60
0.38 50.02 1.93 48.73 11.23 41.03
[C4mim][CF3SO3]
PVA85 0.00 61.17 3.00 57.95 21.06 38.56
0.02 55.28 0.92 53.67 7.40 41.97
PVA30 0.74 48.63 2.84 45.84 23.52 18.36
0.40 52.11 1.39 50.19 29.31 15.74
PVA13 0.59 57.85 2.69 52.38 13.45 24.31
0.78 55.54 1.92 52.23 10.48 27.39
PVA9 1.30 58.72 3.02 58.10 24.49 50.42
1.40 58.54 3.06 57.90 21.13 50.90
3.3.2. Partition of antioxidants
After the characterization of the ABS composed of PVA and the two imidazolium-
based ILs, the capacity of these systems for extracting the antioxidants gallic acid, vanillin,
vanillic acid and syringic acid was evaluated. Therefore a composition was chosen within
the biphasic region of the phase diagrams of the PVA9 and PVA85 with the ILs
[C4mim][BF4] and [C4mim][CF3SO3]: 58 wt. % of IL + 3 wt. % of PVA + 39 wt. % of
antioxidant solution. As mentioned before, for the system composed of PVA85 and
[C4mim][BF4] a different composition was used: 43 wt. % of IL + 2 wt. % of PVA + 55 wt.
87
% of antioxidant solution. In Table 3.4 the average pH values of each phase of the studied
systems are presented; the values were measured after the complete separation of the two
phases, the PVA-rich and the IL-rich phases.
Table 3.4- Average pH values of the PVA-rich (pHPVA) and the IL-rich (pHIL) phase of the ABS used for the
extraction of the antioxidants
Polymer IL pHPVA pHIL
PVA9 [C4mim][BF4] 2.94 ± 0.11 3.75 ± 0.08
[C4mim][CF3SO3] 5.24 ± 0.09 5.67 ± 0.07
PVA85 [C4mim][BF4] 2.03 ± 0.09 2.79 ± 0.12
[C4mim][CF3SO3] 4.99 ± 0.10 5.35 ± 0.14
In Figures 3.12 and 3.13 are represented the average values of the partition coefficient
(K) and the extraction efficiency (EE%), respectively, of the extraction of gallic acid with
the two ILs studied and the PVAs of the highest and the lowest molecular weights (85,000-
124,000 g.mol-1 and 9,000-10,000 g.mol-1). The detailed K and EE% values are presented in
Appendix C.
88
Figure 3.12- Partition coefficients of gallic acid in a biphasic system composed of IL +PVA + antioxidant
solution at 25 °C and average pH values of the IL-rich phase (Bottom) and PVA-rich phase (Top).
0
1
2
3
4
5
6
0.0
0.2
0.4
0.6
0.8
1.0
1.2
C4mimBF4 C4mimCF3SO3
pHK
PVA9 PVA85 pH Top pH Bottom
[C4mim][BF4] [C4mim][CF3SO3]
89
Figure 3.13- Extraction efficiency of gallic acid in a biphasic system composed of IL +PVA + antioxidant
solution at 25 °C and average pH values of the IL-rich phase (Bottom) and PVA-rich phase (Top).
When analyzing the partition of gallic acid between the IL-rich and PVA-rich phases
(Figure 3.12) it can be seen that gallic acid has a slightly higher affinity for the polymer
phase, since K values are below unity, except for [C4mim][BF4] combined with the lighter
PVA. Nevertheless, that is not the case, the K values are that small because the phases have
very different quantities (ca. 0.3g of the PVA-rich phase and ca. 1.7g of the IL-rich phase);
and therefore these values are not much reliable. Considering the EE% that are higher than
50% it is evident that the gallic acid preferentially partitions to the IL-rich phase. As for the
influence of the weight of the polymer, it seems that the high molecular weight polymer has
a lower affinity for the biomolecule than the low molecular weight polymer PVA9, although
the overall data is not as much conclusive. A difficulty when analyzing data stems from the
pH values of the different systems. Although the pH values do not significantly vary between
both phases of the same system, there are significant differences among the systems and
remarkable ones when the IL is changed. Since gallic acid speciation is highly dependent of
the medium pH, this effect may superimpose and even override the intrinsic nature effect.
Comparing the EE% obtained for the gallic acid it is evident that they are higher for the
0
1
2
3
4
5
6
65
70
75
80
85
90
95
C4mimBF4 C4mimCF3SO3
pHEE%
PVA9 PVA85 pH Top pH Bottom
[C4mim][BF4] [C4mim][CF3SO3]
90
PVA9 than for the PVA85 for both ILs. For the systems composed of PVA and
[C4mim][BF4] gallic acid (pKa = 3.94; 9.04; 11.17) [14] is mainly neural, once the pH values
are 2.94 ± 0.11 and 2.03 ± 0.09 for the PVA-rich phase of the PVA9 and PVA85, respectively
and for the IL-rich phase they are 3.75 ± 0.08 and 2.79 ± 0.12 for PVA9 and PVA85,
respectively. But for the systems composed of [C4mim][CF3SO3], gallic acid is mainly
negatively charged. The pH values are 5.24 ± 0.09 and 4.99 ± 0.10 for the PVA-rich phase
for PVA9 and PVA85, respectively, and for the IL-rich phase are 5.67 ± 0.07 and 5.35 ±
0.14 for PVA9 and PVA85, respectively.
The extraction of other antioxidants, namely syringic acid, vanillin and vanillic acid
was also investigated; the partition coefficients as well as extraction efficiency values, were
obtained using ABS composed of PVA9 and [C4mim][CF3SO3]. This system was chosen for
the antioxidant extraction evaluation since it had revealed the best performance in the
extraction of gallic acid.
In the Figures 3.14 and 3.15 the values of K and EE%, respectively, obtained for the
extraction of the four antioxidants along with average pH values of the PVA-rich and IL-
rich phase are represented. The values of pH do not vary significantly among the different
antioxidant systems, which is not surprising due to the comparatively small amount of
antioxidant used.
91
Figure 3.14- Partition coefficients of gallic acid, syringic acid, vanillin and vanillic acid in a biphasic system
composed of 58 wt. % of [C4mim][CF3SO3] + 3 wt. % of PVA9 + 39 wt. % of antioxidant solution at 25 °C
and average pH values of the IL-rich phase (Bottom) and PVA-rich phase (Top).
5.0
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
5.9
6.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
gallic acid syringic acid
pHK
PVA9 pH top pH Bottom
Gallic acid Syringic acid Vanillin Vanillic acid
92
Figure 3.15- Extraction efficiency of gallic acid, syringic acid, vanillin and vanillic acid in a biphasic system
composed of 58 wt. % of [C4mim][CF3SO3] + 3 wt. % of PVA9 + 39 wt. % of antioxidant solution at 25 °C
and average pH values of the IL-rich phase (Bottom) and PVA-rich phase (Top).
The Figures 3.14 and 3.15 show that for both K and EE% increase in the sequence:
gallic acid < syringic acid < vanillin <vanillic acid.
The extraction increases with the increase of the hydrophobicity of the antioxidants
(the log (Kow) values are 0.70, 1.04, 121, and 1.43 for the gallic acid, syringic acid, vanillin
and vanillic acid, respectively [14]). This behaviour was also observed for the extractions of
the same antioxidants with the ABS composed of the IL [P4444]Br and the polymer poly(vinyl
pyrrolidone) (PVP) of 40,000 g.mol-1. In this case, only the vanillin (pKa = 7.81) [14] is
mainly neutral, while the others, gallic acid, vanillic acid (pKa = 4.16; 10.14) [14] and
syringic acid (pKa = 3.93; 9.55) [14], are mainly negatively charged. The EE% obtained
with the ABS composed of [C4mim][CF3SO3] and PVA9 ranges from 90.45% to 95.07%.
These values are higher than those obtained in this work with PVP combined with
phosphonium-based ILs and those obtained in the study by Hasmann et al. [104], that
extracted antioxidants (vanillin and syringic acid) with two polymers (up to 80%). However,
they are not as high as those obtained with some other previously reported studies where it
5.0
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
5.9
6.0
87
88
89
90
91
92
93
94
95
96
97
gallic acid syringic acid
pHEE%
PVA9 pH Top pH Bottom
Gallic acid Syringic acid Vanillin Vanillic acid
93
were obtained EE% up to 99 % (gallic acid, syringic acid and vanillic acid) [123,132]. In
those studies the ABS where composed of ILs and Na2SO4.
94
3.4. Conclusions
This study shows, for the first time, that PVA of four different molecular weights,
9,000-10,000 g.mol-1, 13,000-23,000 g.mol-1, 30,000-70,000 g.mol-1 and 85,000-124,000
g.mol-1 are able to form ABS when combined with the imidazolium-based ILs [C4mim][BF4]
and [C4mim][CF3SO3].
The phase diagrams and respective TLs were determined for these novel ABS formed
by the two imizolium-based ILs combined with four molecular weight PVAs. The results
show that [C4mim][BF4] is a stronger ABS promoter than [C4mim][CF3SO3] when combined
with every PVA studied. Since the two ILs share the same cation, it can be concluded that
the anion of the IL has a strong influence in the ability to form ABS, which agrees with
results from the literature [101].
Also the molecular weight of the PVA influences the ABS forming ability, which
increases when the molecular weight of the polymer increases. This behavior is analogous
to the one observed above for PVP and to the behavior of ABS composed of dextran and
phosphonium-based ILs [147].
The best extraction efficiency value was obtained when using PVA9 combined with
[C4mim][CF3SO3] to extract vanillic acid (95%); this value is close to, although slightly
higher than the one obtained for a ABS composed of the IL [P4444]Br combined with PVP40
(92%). However, there are studies in the literature that obtained higher extraction
efficiencies for the same antioxidants (the gallic acid, vanillic acid and syringic acid) (up to
99%) [123,132], these studies used the inorganic salt Na2SO4 for the formation of the ABS
and have optimized the ABS composition in order to obtain the highest EE%. This
optimization procedure could not (yet) be done in this work, but is meant to be investigated
in the future.
The results here reported evidence that PVA is able to form ABS and to provide
efficient extractions if combined with imidazolium-based ILs. It does not appear to be as
strong two-phase promoter as inorganic salts, but has the advantage of being neutral and
biocompatible.
95
4. Final remarks
96
97
4.1. Conclusions
In this study novel ABS, formed by the combination of hydrophilic polymers, PVP
and PVA, with different families of ionic liquids, were investigated.
The results obtained in this work demonstrated, for the first time, that PVPs of three
different molecular weights, 10,000 g.mol-1, 29,000 g.mol-1, and 40,000 g.mol-1 are able to
form ABS with phosphonium-based ILs. From the ILs studied, [P4444]Br was the strongest
ABS promoter and [P4444]Cl the weakest. Additionally, it was shown that the higher the
molecular weight of the polymer the stronger the ABS forming capability. Regarding the
extractions of the antioxidants, the system with [P4444]Br provided the best extraction
efficiency for syringic acid, vanillin and vanillic acid. For gallic acid the best extraction
efficiency was obtained with [P4441][CH3SO4].
Also for the first time, it was shown that the PVAs of four different molecular weights,
9,000-10,000 g.mol-1, 13,000-23,000 g.mol-1, 30,000-70,000 g.mol-1 and 85,000-124,000
g.mol-1 are able to form ABS combined with the imidazolium-based ILs [C4mim][BF4] and
[C4mim][CF3SO3]. The phase diagrams show that the [C4mim][BF4] is a stronger ABS
promoter than the [C4mim][CF3SO3] whatever the PVAs studied. Concerning the influence
of the molecular weight of PVA, it was shown that the highest molecular weight was the
strongest ABS promoter. This result agrees with the behavior observed above for PVP and
also for other hydrophilic polymers, such as dextran, as already mentioned. The best
extraction efficiency for PVA-based systems was obtained for vanillic acid, 95%, with the
ABS composed of PVA9 and [C4mim][CF3SO3].
4.2. Future work
The experiments carried out during this work lead to new, interesting and promising
results. These results did not provide all the answers but pointed out routes to be followed.
As future work, the parameters that can affect ABS composed of PVP and phosphonium-
based ILs and those composed of PVA and the imidazolium-based ILs, particularly
[C4mim][CF3SO3] and [C4mim][BF4], should be studied in more detail. Among most
relevant parameters in ABS extraction procedures are pH, composition and temperature.
These may alter ABS formation, but most importantly, they may drastically influence the
98
partition of the substances to be extracted by using these systems. A thorough investigation
should then be carried out to evaluate the effect of those parameters on the extraction of the
antioxidants, so as to get an insight on the mechanisms behind these procedures and to
optimize the extraction procedure. A deeper knowledge of the interactions involved among
all players will allow the most adequate choice of the extracting systems according to the
nature of the species being extracted.
Further ahead, it should be interesting to ascertain and/or modulate the selectivity of
the studied ABS by using them in the extraction from a mixture of antioxidants in solution.
Ultimately, the ability of the novel ABS to extract and/or purify antioxidants from a natural
source should be evaluated, which embodies a major step towards greener and more
biocompatible separation processes.
99
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100
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6. Publications
112
113
Co-author in:
Helena Passos, Margarida P. Trindade, Tatiana S.M. Vaz, Luiz P. da Costa, Mara G.
Freire, and João A.P. Coutinho, The impact of self-aggregation on the extraction of
biomolecules in ionic-liquid-based aqueous two-phase systems, Separation and
Purification Technology, 2013, 108, 174-180.
Isabel Boal-Palheiros, Tatiana S. M. Vaz, Mafalda R. Almeida and Mara G. Freire,
Novel aqueous biphasic systems composed of phosphonium-based ionic liquids and
polivinylpirrolidone, 2014, in preparation.
114
115
Appendix A
Calibration curves
116
117
Figure A.1- Calibration curve for gallic acid at 262 nm.
Figure A.2- Calibration curve for vanillin at 280 nm.
Abs = 42.771*[gallic acid]
R² = 0.9980
0.0
0.2
0.4
0.6
0.8
0.000 0.005 0.010 0.015 0.020
Abs
[Gallic acid] / (g.dm-3)
Abs = 68.001*[Vanillin]
R² = 0.9999
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0.000 0.005 0.010 0.015 0.020
Abs
[Vanillin] / (g.dm-3)
118
Figure A.3- Calibration curve for vanillic acid at 292 nm.
Figure A.4- Calibration curve for syringic acid at 274 nm.
Abs = 34.264*[Vanillic acid]
R² = 0.9993
0.0
0.2
0.4
0.6
0.8
1.0
0.000 0.005 0.010 0.015 0.020 0.025 0.030
Abs
[Vanillic acid] / (g.dm-3)
Abs = 41.817*[Syringic acid]
R² = 0.9988
0.0
0.2
0.4
0.6
0.8
0.000 0.005 0.010 0.015 0.020
Abs
[Syringic acid] / (g.dm-3)
119
Appendix B
Experimental binodal data
120
121
Table B.1- Experimental weight fraction data for the systems composed of IL + PVP40 + H2O at 25
°C and at atmospheric pressure.
[P4444]Br [Pi(444)1][Tos]
Mw = 339.33 g.mol-1 Mw = 384.47 g.mol-1
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
31.931 28.285 14.137 45.017 16.767 62.680 6.237 72.639
30.784 28.861 13.567 45.843 14.911 64.501 5.890 73.172
29.301 30.141 12.929 46.574 13.540 65.554 5.527 73.657
28.082 31.199 12.441 47.468 12.308 66.647 5.069 74.186
26.368 32.881 11.884 47.857 11.296 67.401 4.712 74.536
25.333 33.493 11.437 48.473 10.164 68.820 4.403 74.780
24.197 34.648 11.034 49.318 9.465 69.739 4.089 75.324
23.287 35.443 10.544 49.519 8.490 70.310 3.883 75.362
22.425 36.251 10.158 50.314 7.955 70.964 3.539 75.981
21.646 36.988 9.808 50.571 7.485 71.527 3.219 76.526
20.533 38.172 9.469 50.861 7.097 71.990 3.096 76.401
19.667 39.091 8.958 51.534 6.463 72.616
18.959 39.649 8.537 51.996
18.070 40.681 8.161 52.883
17.189 41.554 7.733 53.047
16.520 42.341 7.172 53.616
15.917 43.310 6.847 54.188
15.300 44.174 6.497 54.880
14.606 44.501 6.119 55.239
122
Table B.2- Experimental weight fraction data for the systems composed of IL + PVP40 + H2O at 25
°C and at atmospheric pressure.
[P4441][CH3SO4]
Mw = 328.45 g.mol-1
[P4442][Et2PO4]
Mw = 384.47 g.mol-1
[P4444]Cl
Mw = 294.88 g.mol-1
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
16.323 57.637 16.735 62.751 12.895 63.056
14.955 58.910 15.545 62.961 11.321 63.704
13.035 61.369 14.447 64.115 10.458 64.090
12.146 61.999 13.238 65.134 9.949 64.719
11.420 62.881 12.498 65.568 8.769 65.879
10.371 63.928 11.817 65.942 7.690 66.656
9.568 64.860 11.110 66.466 6.907 67.577
8.887 65.508 10.356 67.066 6.408 67.757
8.251 66.131 9.650 67.820 6.038 68.059
7.915 66.694 9.066 68.298 5.688 68.331
7.570 66.743 8.400 69.010 5.263 68.701
7.129 67.237 7.891 69.400 4.964 69.181
7.455 69.929 4.697 69.179
4.409 69.572
4.067 70.100
Table B.3- Experimental weight fraction data for the systems composed of IL + PVP29 + H2O at 25
°C and at atmospheric pressure.
[P4441][CH3SO4]
Mw = 328.45 g.mol-1
[P4442][Et2PO4]
Mw = 384.47 g.mol-1
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
21.433 54.113 19.670 63.413 8.754 71.100
19.819 54.979 17.437 65.050 8.302 71.531
16.137 59.005 15.918 66.015 7.951 71.642
12.389 63.614 14.486 67.517 7.504 72.125
11.424 64.259 13.542 68.165 7.059 72.709
10.175 65.478 13.027 67.899 6.718 73.043
12.071 68.622 6.407 73.195
11.227 69.341 6.201 73.361
10.490 70.011 5.869 73.625
9.979 70.070 5.625 73.853
9.394 70.575
123
Table B.4- Experimental weight fraction data for the systems composed of IL + PVP29 + H2O at 25
°C and at atmospheric pressure.
[Pi(444)1][Tos] [P4444]Br
Mw = 384.47 g.mol-1 Mw = 339.33 g.mol-1
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
18.150 61.535 36.217 28.600 15.382 47.222
14.774 65.607 32.887 30.205 14.196 48.588
12.462 67.481 31.563 31.108 13.338 49.644
11.536 68.167 29.532 32.983 12.475 50.536
10.487 69.194 27.922 34.601 11.875 51.206
9.581 70.068 24.682 37.044 11.261 52.004
9.120 70.483 22.442 39.281 10.750 52.468
8.324 71.287 20.607 41.313 10.133 53.227
7.642 71.945 18.913 43.232
7.127 72.624 17.374 45.057
6.683 73.101 16.590 45.855
124
Table B.5- Experimental weight fraction data for the systems composed of IL + PVP10 + H2O at 25
°C and at atmospheric pressure.
[P4441][CH3SO4]
Mw = 328.45
g.mol-1
[P4442][Et2PO4]
Mw = 384.47
g.mol-1
[Pi(444)1][Tos] [P4444]Br
Mw = 384.47
g.mol-1
Mw = 339.33
g.mol-1
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
14.137 63.815 10.055 82.171 7.631 82.211 36.968 30.191
12.072 65.299 8.409 84.989 6.527 82.981 33.212 33.352
10.674 66.257 3.627 93.569 4.790 85.217 30.920 34.893
9.146 67.523 2.940 94.752 27.892 38.163
7.512 69.579 26.664 39.143
5.189 72.770 23.995 42.187
4.344 73.249 21.662 44.861
3.786 74.232 20.146 46.501
18.132 48.964
16.638 50.685
15.474 52.072
14.336 53.441
13.054 54.985
12.116 56.198
11.140 57.366
10.543 58.268
10.033 58.701
9.501 59.284
8.845 60.091
125
Table B.6- Experimental weight fraction data for the systems composed of [C4mim][BF4] + PVA +
H2O at 25 °C and at atmospheric pressure.
PVA85 PVA30 PVA13 PVA9
Mw = 85,000-
124,000
Mw = 30,000-
70,000
Mw = 13,000-
23,000 Mw = 9,000-10,000
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
2.055 34.664 2.740 33.491 3.112 36.075 5.021 43.662
1.993 34.667 1.598 34.542 2.867 36.223 4.468 43.816
1.881 34.791 1.413 34.870 2.660 36.384 4.303 43.944
1.783 34.803 1.177 35.609 2.306 36.649 3.731 44.159
1.690 34.939 0.938 35.973 1.873 37.023 3.201 44.593
1.608 35.068 0.837 36.150 1.765 37.232 2.718 45.027
1.358 35.326 0.709 36.816 1.505 37.800 2.490 45.240
1.261 35.417 0.603 37.673 1.364 37.985 2.291 45.430
1.129 35.682 0.521 38.835 1.157 38.549 2.248 45.475
1.008 35.784 1.068 38.716 2.224 45.531
0.877 36.032 1.010 38.992 2.163 45.644
0.563 36.774 0.873 39.345 2.110 45.683
0.485 37.230 1.942 45.922
1.778 46.322
1.682 46.399
1.616 46.392
1.613 46.583
1.552 46.645
1.482 46.753
1.427 46.900
1.380 46.955
1.376 46.984
1.314 47.178
1.051 48.125
0.985 48.302
0.825 49.513
0.761 49.908
126
Table B.7- Experimental weight fraction data for the systems composed of [C4mim][CF3SO3] + PVA
+ H2O at 25 °C and at atmospheric pressure.
PVA85 PVA30 PVA13 PVA9
Mw = 85,000-
124,000
Mw = 30,000-
70,000
Mw = 13,000-
23,000 Mw = 9,000-10,000
100 w1 100 w2 100 w1 100 w2 100 w1 100 w2 100 w1 100 w2
1.699 46.019 2.300 41.219 2.166 45.970 3.551 56.078
1.607 46.123 1.990 41.931 2.110 46.055 3.450 56.196
1.514 46.261 1.916 42.428 1.943 46.708 3.129 56.429
1.442 46.397 1.680 43.406 1.855 47.092 2.949 56.692
1.304 46.672 1.621 43.659 1.664 48.017 2.805 56.770
1.248 46.722 1.362 44.738 1.529 49.213 2.482 57.233
1.193 46.815 1.064 46.428 1.355 50.765 2.465 57.268
1.175 46.917 0.684 49.163 2.366 57.328
1.106 47.062 2.206 57.457
0.775 48.020 2.119 57.635
0.569 48.431 1.931 57.862
0.515 48.809 1.852 57.915
1.664 57.971
1.352 58.318
1.286 58.532
1.216 58.573
1.145 58.851
127
Appendix C
Experimental data for
partitioning of antioxidants
128
129
In the following Figures C.1 to C.4 are presented the speciation diagrams that shows
the effect of the pH in the concentration of the different molecular species that can exist in
aqueous solutions of gallic acid, vanillin, vanillic acid and syringic acid.
Figure C.1-Speciation diagram of gallic acid [14].
0
20
40
60
80
100
0 2 4 6 8 10 12 14
Con
cen
trat
ion
(w
t.%
)
pH
Gallic acid vanillic acid vanillin syrinic acidsyringic acidGallic acid vanillic acid vanillin syrinic acidsyringic acid
-
Gallic acid vanillic acid vanillin syrinic acidsyringic acid
-
- -
Gallic acid vanillic acid vanillin syrinic acidsyringic acid
-
-
Gallic acid vanillic acid vanillin syrinic acidsyringic acid
-
-
Gallic acid vanillic acid vanillin syrinic acidsyringic acid
-
- -
130
Figure C.2- Speciation diagram of vanillin [14].
Figure C.3- Speciation diagram of vanillic acid [14].
0
20
40
60
80
100
0 2 4 6 8 10 12 14
Con
cen
trat
ion
(w
t. %
)
pH
0
20
40
60
80
100
0 2 4 6 8 10 12 14
Con
cen
trat
ion
(w
t. %
)
pH
Gallic acid vanillic acid vanillin syrinic acidsyringic acidGallic acid vanillic acid vanillin syrinic acidsyringic acid
-
Gallic acid vanillic acid vanillin syrinic acidsyringic acidGallic acid vanillic acid vanillin syrinic acidsyringic acid
-
Gallic acid vanillic acid vanillin syrinic acidsyringic acid
-
-
131
Figure C.4- Speciation diagram of syringic acid [14].
0
20
40
60
80
100
0 2 4 6 8 10 12 14
Con
cen
trat
ion
(w
t. %
)
pH
Gallic acid vanillic acid vanillin syrinic acidsyringic acidGallic acid vanillic acid vanillin syrinic acidsyringic acid
-
Gallic acid vanillic acid vanillin syrinic acidsyringic acid
-
-
132
In Tables C.1 to C.2 the weight fraction percentages of the ILs and PVPs at the
extraction points are presented, as well as the respective partition coefficients and extraction
efficiencies of the different antioxidants. In Table C.3 the correspondent values for the
systems composed of ILs and PVAs are shown.
Table C.1- Weight fraction percentage of the IL and PVP in the mixture and the partition coefficients
(K) and extraction efficiency (EE%) of the extractions of the antioxidants gallic acid, vanillin, vanillic acid
and syringic acid with PVP40.
IL Antioxidant
Weight fraction
percentage (wt. %) EE% K
[IL]M [PVP]M
[P4444]Br
Gallic acid 66.99 13.06 73.556 ± 0.294 1.262 ± 0.057
Vanillin 66.60 12.96 86.735 ± 0.285 2.543 ± 0.324
Vanillic acid 66.81 13.11 91.998 ± 0.039 4.241 ± 0.058
Syringic acid 66.94 13.10 84.223 ± 1.604 1.909 ± 0.199
[P4441][CH3SO4]
Gallic acid 66.82 12.99 81.403 ± 0.091 2.472 ± 0.124
Vanillin 66.76 13.05 71.967 ± 1.616 1.382 ± 0.057
Vanillic acid 66.76 13.02 71.221 ± 0.247 1.240 ± 0.149
Syringic acid 66.85 12.94 74.937 ± 1.135 1.662 ± 0.058
[P4442][Et2PO4]
Gallic acid 66.71 12.98 74.178 ± 1.047 1.253 ± 0.052
Vanillin 66.98 12.99 75.931 ± 1.324 1.368 ± 0.033
Vanillic acid 67.00 12.71 81.802 ± 1.082 1.701 ± 0.101
Syringic acid 66.84 13.12 73.985 ± 1.668 1.254 ± 0.127
[Pi(444)1][Tos]
Gallic acid 66.74 13.01 80.365 ± 0.020 2.048 ± 0.252
Vanillin 66.83 12.92 74.037 ± 2.504 1.708 ± 0.194
Vanillic acid 66.70 12.97 69.335 ± 2.016 1.420 ± 0.314
Syringic acid 67.02 13.02 77.100 ± 1.269 1.959 ± 0.208
[P4444]Cl
Gallic acid 66.48 13.25 68.289 ± 1.202 0.979 ± 0.011
Vanillin 66.78 13.05 74.511 ± 0.071 1.430 ± 0.095
Vanillic acid 66.75 12.95 78.510 ± 2.261 1.503 ± 0.056
Syringic acid 66.96 12.98 67.992 ± 0.306 0.995 ± 0.034
133
Table C.2- Weight fraction percentage of the IL and PVP in the mixture and the partition coefficients
(K) and extraction efficiency (EE%) of the extractions of gallic acid, vanillin, vanillic acid and syringic acid
with [P4444]Br.
Polymer Antioxidant
Weight fraction
percentage (wt. %) EE% K
[IL]M [PVP]M
PVP29
Gallic acid 66.83 12.96 80.739 ± 0.567 1.454 ± 0.017
Vanillin 66.86 12.99 89.870 ± 0.762 3.112 ± 0.007
Vanillic acid 66.51 13.07 84.943 ± 0.950 2.030 ± 0.082
Syringic acid 66.90 13.03 83.721 ± 0.402 1.875 ± 0.036
PVP10
Gallic acid 66.71 12.99 89.204 ± 0.167 2.180 ± 0.158
Vanillin 66.86 13.08 91.464 ± 0.459 3.179 ± 0.044
Vanillic acid 66.99 13.02 80.781 ± 1.712 1.226 ± 0.150
Syringic acid 66.81 12.93 83.337 ± 0.724 1.417 ± 0.011
Table C.3- Weight fraction percentage of the IL and PVA in the mixture and the partition coefficients
(K) and extraction efficiency (EE%) of the extractions of gallic acid, vanillin, vanillic acid and syringic acid
with [C4mim][CF3SO3] and of the extraction of gallic acid with [C4mim][BF4].
IL + PVA + H2O Antioxidant
Weight fraction
percentage (wt.
%) EE% K
[IL]M [PVA]M
[C4mim][CF3SO3]
PVA9
Gallic acid 57.95 3.07 90.450 ± 0.492 0.909 ± 0.003
Vanillin 57.97 3.01 93.051 ± 0.179 1.337 ± 0.053
Vanillic acid 57.90 3.05 95.074 ± 0.517 1.554 ± 0.198
Syringic acid 57.97 3.07 92.801 ± 0.237 1.098 ± 0.026
PVA85 Gallic acid 58.08 0.03 79.161 ± 0.203 0.943 ± 0.053
[C4mim][BF4]
PVA9 Gallic acid 58.00 3.04 86.375 ± 0.016 0.894 ± 0.050
PVA85 Gallic acid 43.24 2.23 83.784 ± 0.213 1.121 ± 0.008