tatiana sofia marçalo vaz bifásicos extraction of ... de antioxidantes com... · problemas devido...

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

I

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.

III

Aos meus pais e ao meu maninho...

V

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

VII

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.

IX

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.

XI

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. .

XIII

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

XIV

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

XV

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

XVII

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


°C in mol.kg-1 units (left) and in wt.% (right): ▲, [P4444]Br; ♦, [P4441][CH3SO4]; ■,

[P4442][Et2PO4]; ▲, [Pi(444)1][Tos]. ...................................................................................... 51


°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

XVIII

Figure 2.9- Phase diagrams for ternary systems composed of [P4442][Et2PO4] + PVP + H2O


Figure 2.10- Phase diagrams for ternary systems composed of [Pi(444)1][Tos] + PVP + H2O


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 3.10- 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


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


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


XXI

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;



PVAc - Poly(vinyl acetate);

PVP - Poly(vinyl pyrrolidone);

PVP10 - Poly(vinyl pyrrolidone) of 10,000 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;

XXIV

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;

XXV

[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.

1

1. General introduction

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

mb

er o

f p

ub

lish

ed

arti

cle

s

Year

28

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.

35

2. IL + PVP + H2O ABS

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.


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.


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


Figure 2.10- Phase diagrams for ternary systems composed of [Pi(444)1][Tos] + PVP + H2O at 25 °C in


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


pHEE%

PVP40 PVP29 PVP10 pH Top pH Bottom


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.

71

3. IL + PVA + H2O ABS

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





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


Figure 3.11- Phase diagram for the ternary system composed of [C4mim][CF3SO3] + PVA13 + H2O: binodal


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


pHK

PVA9 pH top pH Bottom


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


pHEE%

PVA9 pH Top pH Bottom


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

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

5. References

101

1. Carocho, M., Ferreira, I.C.F.R. A review on antioxidants, prooxidants and related

controversy: natural and synthetic compounds, screening and analysis methodologies and

future perspectives. Food and Chemical Toxicology, 2013. 51: p. 15–25.

2. Kochanek, K.S., Brenneisen, P., Wenk, J., Herrmann, G., Ma, W., Kuhr, L., Meewes,

C., Wlaschek, M., Photoaging of the skin from phenotype to mechanisms. Experimental

Gerontology, 2000. 35: p. 307–316.

3. Belter, P.A., Cussler, E.L., Hu, W.S., Separation and Purification Reviews. Wiley,

1988. 17(2): p. 207–208.

4. Luque de Castro, M.D., Priego-Capote, F., Soxhlet extraction: Past and present

panacea. Journal of chromatography A, 2010. 1217(16): p. 2383–2389.

5. Martínez-Aragón, M., Burghoff, S., Goetheer, E.L.V., de Haan, A.B., Guidelines for

solvent selection for carrier mediated extraction of proteins. Separation and Purification

Technology, 2009. 65(1): p. 65–72.

6. Albertsson, P.A., Partition of proteins in liquid polymer-polymer two-phase systems.

Nature, 1958. 182(4637): p. 709-711.

7. Luo, J., Conrad, O., Vankelecom, I.F.J., Physicochemical properties of

phosphonium-based and ammonium-based protic ionic liquids. Journal of Materials

Chemistry, 2012. 22(38): p. 20574–20579.

8. Restolho, J., Mata, J.L., Saramago, B., Choline based ionic liquids: Interfacial

properties of RTILs with strong hydrogen bonding. Fluid Phase Equilibria, 2012. 322(0): p.

142–147.

9. Halliwell, B., Gutteridge, J.M.C., Free Radicals in Biology and Medicine Second

Edition. 1989.

10. Sies, H., Physiological society symposium: impaired endothelial and smooth muscle

cell function in oxidative stress; oxidative stress: oxidants and antioxidants. Experimental

Physiology, 1996. 82: p. 291–295.

11. Pisoschi, A.M., Negulescu, G.P., Methods for Total Antioxidant Activity

Determination: A Review. Biochemistry and Analytical Biochemistry, 2012. 1(1): p. 1–10.

12. Sanghera, G.S., Malhotra, P.K., Sidhu, G.S., Sharma, V.K., Sharma, B.B., Karan, R.,

Genetic Engineering of Crop Plants for Enhanced Antioxidants Activity. International

Journal of Advancements in Research and Technology, 2013. 2(5): p. 428–458.

13. Pietta, P.G., Flavonoids as antioxidants. Journal of natural products, 2000. 63(7): p.

1035–1042.

14. ChemSpider - The free chemical database. 2014 [cited 2014 June]; Available from:

www.chemspider.com.

15. Koyama, A., Bitsch, I., Pharmacokinetics of Gallic Acid and Its Relative

Bioavailability from Tea in Healthy Humans. Human Nutrition and Metabolism, 2001. 1: p.

1207–1210.

16. Aruoma, O.I., Murcia, A., Butler, J., Halliwell, B., Evaluation of the antioxidant and

prooxidant actions of gallic acid and its derivatives. Journal of Agricultural and Food

Chemistry, 1993. 41(11): p. 1880–1885.

17. Kroes, B.H., van den Berg, A.J., Quarles van Ufford, H.C., van Dijk, H., Labadie,

R.P., Anti-inflammatory activity of gallic acid. Planta Medica, 1992. 58(6): p. 499–504.

18. Chuang, C.Y., Liu, H.C., Wu, L.C., Chen, C.Y., Chang, J.T., Hsu, S.L., Gallic acid

induces apoptosis of lung fibroblasts via a reactive oxygen species-dependent ataxia

telangiectasia mutated-p53 activation pathway. Journal of agricultural and food chemistry,

2010. 58(5): p. 2943–2951.

102

19. Abdelwahed, A., Bouhlel, I., Skandrani, I., Valenti, K., Kadri, M., Guiraud, P.,

Steiman, R., Mariotte, A., Ghedira, K., Laporte, F., Dijoux-Franca, M., Chekir-Ghedira, L.,

Study of antimutagenic and antioxidant activities of gallic acid and 1,2,3,4,6-

pentagalloylglucose from Pistacia lentiscus. Confirmation by microarray expression

profiling. Chemico-biological interactions, 2007. 165(1): p. 1–13.

20. Liu, Z., Schwimer, J., Liu, D., Lewis, J., Greenway, F.L., York, D.A., Woltering,

E.A., Gallic Acid is Partially Responsible for the Antiangiogenic Activities of Rubus Leaf

Extract. Phytotherapy research, 2006. 813: p. 806–813.

21. Boyd, I., Beveridge, E.G., Relationship between the anti-bacterial activity towards

escerichia-coli NCTC 5933 and the physicochemical properties of some esters of 3,4,5,-

trihydroxybenzoic acid (Gallic acid). Microbios, 1979. 24(97): p. 173–184.

22. Ban, J.Y., Nguyen, H.T.T., Lee, H.J., Cho, S.O., Ju, H.S., Kim, J.Y., Bae, K., Song,

K.S., Seong, Y.H., Neuroprotective properties of gallic acid from Sanguisorbae radix on

amyloid beta protein (25--35)-induced toxicity in cultured rat cortical neurons. Biological

and pharmaceutical bulletin, 2008. 31(1): p. 149–153.

23. Sohi, K.K., Mittal, N., Hundal, M.K., Khanduja, K.L., Gallic acid, an antioxidant,

exhibits antiapoptotic potential in normal human lymphocytes: A Bcl-2 independent

mechanism. Journal of nutritional science and vitaminology, 2003. 49(4): p. 221–227.

24. Nakano, N., Shirasaka, N., Koyama, H., Hino, M., Murakami, T., Shimizu, S.,

Yoshizumi, H., C19 Odd-chain Polyunsaturated Fatty Acids (PUFAs) are metabolized to

C12-PUFAs in a rat liver cell line, and curcumin, gallic acid, and their related compounds

inhibit their desaturation. Bioscience, Biotechnology, and Biochemistry, 2000. 64(8): p.

1641–1650.

25. Sanae, F., Miyaichi, Y., Hayashi, H., Potentiation of vasoconstrictor response and

inhibition of endothelium-dependent vasorelaxation by gallic acid in rat aorta. Planta

Medica, 2002. 68(8): p. 690–693.

26. Inoue, M., Suzuki, R., Sakaguchi, N., Li, Z., Takeda, T., Ogihara, Y., Jiang, B.Y.,

Chen, Y., Selective Induction of Cell Death in Cancer Cells by Gallic acid. Biological and

pharmaceutical bulletin, 1995. 18: p. 1526–1530.

27. Madlener, S., Illmer, C., Horvath, Z., Saiko, P., Losert, A., Herbacek. I., Grusch, M.,

Elford, H.L., Krupitza, G., Bernhaus, A., Fritzer-Szekeres, M., Szekeres, T., Gallic acid

inhibits ribonucleotide reductase and cyclooxygenases in human HL-60 promyelocytic

leukemia cells. Cancer Letters, 2007. 245(1): p. 156–162.

28. Sinha, A.K., Sharma, U.K., Sharma, N., A comprehensive review on vanilla flavor:

extraction, isolation and quantification of vanillin and others constituents. International

journal of food sciences and nutrition, 2008. 59(4): p. 299–326.

29. Sostaric, T., Boyce, M.C., Spickett, E.E., Analysis of the Volatile Components in

Vanilla Extracts and Flavorings by Solid-Phase Microextraction and Gas Chromatography.

Journal of Agricultural and Food Chemistry, 2000. 48(12):p. 5802–5807.

30. Dhananjaya, B.L., Nataraju, A., Rajesh, R., Gowda, C.D.R., Sharath, B.K.,

Vishwanath, B.S., Anticoagulant effect of Naja naja venom 5´nucleotidase : Demonstration

through the use of novel specific inhibitor, vanillic acid. Toxicon, 2006. 48: p. 411–421.

31. Dhananjaya, B.L., Nataraju, A., Raghavendra, Gowda, C.D., Sharath, B.K., D’Souza,

C.J., Vanillic acid as a novel specific inhibitor of snake venom 5’-nucleotidase: a

pharmacological tool in evaluating the role of the enzyme in snake envenomation.

Biochemistr, 2009. 74(12): p. 1315–1319.

103

32. Vetrano, A.M., Heck, D.E., Thomas, M., Mishin, V., Laskin, D.L., Laskin, J.D.,

Mariano, T.M., Characterization of the Oxidase Activity in Mammalian Catalase *. The

Journal of Biological Chemistry, 2005. 280(40): p. 35372–35381.

33. Huang, S., Hsu, C., Chuang, H., Shih, P., Wu, C., Yen, G., Inhibitory effect of vanillic

acid on methylglyoxal-mediated glycation in apoptotic Neuro-2A cells. Neurotoxicology,

2008. 29: p. 1016–1022.

34. Huang, S.M., Chuang, H.C., Wu, C.H., Yen, G.C., Cytoprotective effects of phenolic

acids on methylglyoxal-induced apoptosis in Neuro-2A cells. Molecular Nutrition and Food

Research, 2008. 52(8): p. 940–949.

35. Toh, A.I., Soda, K.I., Ondoh, M.K., Awase, M.K., Obayashi, M.K., Hepatoprotective

Effect of Syringic Acid and Vanillic Acid on Concanavalin A-Induced Liver Injury.

Biological and pharmaceutical bulletin, 2009. 32(7): p. 1215–1219.

36. Teissedre, P.L., Waterhouse, L., Inhibition of oxidation of human low-density

lipoproteins by phenolic substances in different essential oils varieties. Journal of

agricultural and food chemistry, 2000. 48(9): p. 3801–3805.

37. Burri, J., Graf, M.P., Lambelet, P., Loeliger, J., Protection of a food product against

oxydation. European Patent 0,340,500. 1989.

38. Fitzgerald, D.J., Stratford, M., Gasson, M.J., Ueckert, J., Bos, A., Narbad, A., Mode

of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and

Listeria innocua. Journal of applied microbiology, 2004. 97(1): p. 104–113.

39. Akagia, K., Hirose, M., Hoshiyaa, T., Mizoguchia, Y., Shiraia, T., Modulating effects

of ellagic acid , vanillin and quercetin in a rat medium term multi-organ carcinogenesis

model. Cancer Letter, 1995. 94: p. 113–121.

40. Ong, L.X.S., Ang, M.W., Peng, X.U., Ang, Y.Y., Qiang, Z., Hang, Z., Experimental

and Theoretical Studies on the Inclusion Complexation of Syringic Acid with alpha-, betha-

, gama- and Heptakis(2,6-di-O-methyl)-betha-cyclodextrin. Chemical and pharmaceutical

bulletin, 2008. 56(4): p. 468–474.

41. Hirota, A., Taki, S., Kawaii, S., Yano, M., Abe, N., 1, 1-Diphenyl-2-picrylhydrazyl

radical-scavengi ng compounds from soybean misoand antiprol iferative activity of

isoflavones from soybean misotoward the cancer cell lines. Bioscience, Biotechnology, and

Biochemistry, 2000. 64(5): p. 1038–1040.

42. Yunhai, L., Jianguo, F., Ting, L., Wenqing, W., Aihua, L., Anti-endotoxic effects of

syringic acid of Radix Isatidis. Journal of Huazhong University of Science and Technology,

2003. 23(2): p. 206–208.

43. Guimaraes, C.M., Giao, M.S., Martinez, S.S., Pintado, A.I., Pintado, M.E., Bento,

L.S., Antioxidant Activity of Sugar Molasses, Including Protective Effect Against DNA

Oxidative Damage. Journal of Food Science, 2007. 72(1): p. 39-43.

44. Rinne, T., Mutschler, E., Wimmer-Greinecker, G., Moritz, A., Olbrich, H.G.,

Vitamins C and E protect isolated cardiomyocytes against oxidative damage. International

journal of cardiology, 2000. 75(2-3): p. 275–281.

45. Niki, E., Noguchi, N., Tsuchihashi, H., Gotoh, N., Interaction among vitamin C ,

vitamin E , and ß-carotene. The American Journal of Clinical Nutrition, 1995. 62: p. 1322-

1326.

46. Chevion, S., Roberts, M.A., Chevion, M., The use of cyclic voltammetry for the

evaluation of antioxidant capacity. Free Radical Biology and Medicine, 2000. 28(6): p. 860–

870.

47. Antolovich, M., Prenzler, P.D., Patsalides, E., McDonald, S., Robards, K., Methods

for testing antioxidant activity. Analyst, 2002. 127(1): p. 183–198.

104

48. Kanner, J., German, J.B., Kinsella, J.E., Hultin, H.O., Initiation of lipid peroxidation

in biological systems. Food Science and Nutrition, 1987. 25(4): p. 317–364.

49. Cheeseman, K.H., Slater, T.F., An introduction to free radical biochemistry. British

Medical Bulletin, 1993. 49(3): p. 481–493.

50. Madhavi, D.L., Deshpande, S.S., Salunkhe, D.K., Food Antioxidants: Technological:

Toxicological and Health Perspectives. Taylor & Francis; 1995.

51. Frankel, E.N., Meyer, A.S., The problems of using one-dimensional methods to

evaluate multifunctional food and biological antioxidants. Journal of the Science of Food

and Agriculture, 2000. 80(13): p. 1925–1941.

52. Harman, D., The Free Radical Theory of Aging. Antioxidants and Redox Signaling,

5th ed. 2003. p. 557–561.

53. Pansarasa, O., Castagna, L., Colombi, B., Vecchiet, J., Felzani, G., Marzatico, F.,

Age and sex differences in human skeletal muscle: Role of reactive oxygen species. Free

Radical Research, 2000. 33(3): p. 287–293.

54. Fanò, G., Mecocci, P., Vecchiet, J., Belia, S., Fulle, S., Polidori, M.C., Felzani, G.,

Senin, U., Vecchiet, L., Beal, M.F., Age and sex influence on oxidative damage and

functional status in human skeletal muscle. Journal of Muscle Research & Cell Motility,

2001. 22(4): p. 345–351.

55. Cesari, M., Pahor, M., Bartali, B., Cherubini, A., Penninx, B.W.J.H., Williams, G.R.,

Atkinson, H., Martin, A., Guralnik, J.M., Ferrucci, L., Antioxidants and physical

performance in elderly persons: the Invecchiare in Chianti study. The American Journal of

Clinical Nutrition, 2004. 79(2): p. 289–294.

56. Harman, D., Aging: A Theory Based on Free Radical and Radiation Chemistry.

Journal of Gerontology, 1956. 11(3): p. 298–300.

57. Harman, D., The biologic clock: the mitochondria? Journal of the American

Geriatrics Society, 1972. 20(4): p. 145–147.

58. Petersen, K.F., Befroy, D., Dufour, S., Dziura, J., Rothman, D.L., Dipietro, L., Cline,

G.W., Gerald, I., Mitochondrial Dysfunction in the Elderly: Possible Role in Insulin

Resistance. National Institute of Health, 2003. 300(5622): p. 1140–1142.

59. Goswami, A., Dikshit, P., Mishra, A., Mulherkar, S., Nukina, N., Jana, N.R.,

Oxidative stress promotes mutant huntingtin aggregation and mutant huntingtin-dependent

cell death by mimicking proteasomal malfunction. Biochemical and Biophysical Research

Communications, 2006. 342(1): p. 184–190.

60. Halvorsen, B.L., Carlsen, M.H., Phillips, K.M., Bøhn, S.K., Holte, K., Jacobs, D.R.,

Blomhoff, R., Content of redox-active compounds (ie, antioxidants) in foods consumed in

the United States. The American Journal of Clinical Nutrition, 2006. 84(1): p. 95–135.

61. Serrano-Pozo, A., Frosch, M.P., Masliah, E., Hyman, B.T., Neuropathological

alterations in Alzheimer disease. Cold Spring Harbor perspectives in medicine, 2011. 1(1):

p. 1–23.

62. Gella, A., Durany, N., Oxidative stress in Alzheimer disease. Cell Adhesion and

Migration, 2009. 3(1): p. 88–93.

63. Bekris, L.M., Millard, S., Lutz, F., Li, G., Galasko, D.R., Martin, R., Quinn, J.F.,

Kaye, J.A., Leverenz, J.B., Debby, W., Yu, C., Peskind, E.R., Tau Phosphorylation Pathway

Genes and Cerebrospinal Fluid Tau Levels in Alzheimer’s Disease. National Institute of

Health, 2012. 0(7): p. 874–883.

64. Sano, M., Ernesto, C., Thomas, R.G., Klauber, M.R., A controlled trial af selegiline,

alpha-tocopherol, or both as treatment for Alzheimer´s disease. The New England Journal

of Medicine, 2000. 336: p. 1216-1222.

105

65. Morris, M.C., Evans, D.A., Bienias, J.L., Tangney, C.C., Bennett, D.A., Wilson,

R.S., Scherr, P.A., Dietary Intake of Antioxidant Nutrients and the Risk of Incident Alzheimer

Disease in a Biracial Community Study. The Journal of the American Medical Association,

2002. 287(24): p. 3230-3237.

66. Amidon, G., Lennernäs, H., Shah, V.P., Crison, J.R., A Theoretical Basis for a

Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution

and in Vivo Bioavailability. Pharmaceutical Research, 1995. p. 413–420.

67. Thompson, T.N., Early ADME in Support of Drug Discovery: The Role of Metabolic

Stability Studies. Current Drug Metabolism, 2000. p. 215–241.

68. Zhang, L., Zheng, Y., Chow, M.S.S., Zuo, Z., Investigation of intestinal absorption

and disposition of green tea catechins by Caco-2 monolayer model. International Journal of

Pharmaceutics, 2004. 287: p. 1–12.

69. Viscovich, M., Lykkesfeldt, J., Poulsen, H.E., Vitamin C pharmacokinetics of plain

and slow release formulations in smokers. Clinical Nutrition, 2004. 23(5): p. 1043–1050.

70. Gilgun-Sherki, Y., Melamed, E., Offen, D., Oxidative stress induced-

neurodegenerative diseases: the need for antioxidants that penetrate the blood brain barrier.

Neuropharmacology, 2001. 40(8): p. 959–975.

71. Ratnam, D.V., Ankola, D.D., Bhardwaj, V., Sahana, D.K., Kumar, M.N.V.R., Role

of antioxidants in prophylaxis and therapy: A pharmaceutical perspective. Journal of

controlled release, 2006. 113(3): p. 189–207.

72. Kommuru, T.R., Gurley, B., Khan, M.A., Reddy, I.K., Self-emulsifying drug delivery

systems (SEDDS) of coenzyme Q10: formulation development and bioavailability

assessment. International journal of pharmaceutics, 2001. 212(2): p. 233–246.

73. Lewis II J., DiNardo J.C., McDaniel D.H., Oxidative Stress, the Damage-

Accumulation Theory of Skin Aging, and the Role of Antioxidants in the Future of Topical

Skin Protection. Cosmetic Dermatology, 2009. 22(11): p. 576–582.

74. Nishigori, C., Hattori, Y., Toyokuni, S., role of reactive oxygen species in skin

carcinogenesis. Antioxidants and Redox Signaling, 2004. 6(3): p. 561–570.

75. Reelfs, O., Tyrrell, R.M., Pourzand, C., Ultraviolet A Radiation-Induced Immediate

Iron Release Is a Key Modulator of the Activation of NF- j B in Human Skin Fibroblasts.

Journal of Investigative Dermatology 2004. 122: p. 1440–1447.

76. Mariéthoz, E., Richard, M., Polla, L.L., Kreps, S.E., Dal'Ava, J., Polla B.S.,

Oxidant/antioxidant imbalance in skin aging: environmental and adaptive factors. Reviews

on Environmental Health, 1998. 13(3): p. 147–168.

77. Brosche, T., Platt, D., Effect of borage oil consumption on fatty acid metabolism,

transepidermal water loss and skin parameters in elderly people. Archives of gerontology

and geriatrics, 2000. 30(2): p. 139–150.

78. Boelsma, E., Hendriks, H.F.J., Roza, L., Nutritional skin care : health effects of

micronutrients and fatty. The American Journal of Clinical Nutrition, 2001. 73(1): p. 853–

864.

79. Toh, A.I., Soda, K.I., Ondoh, M.K., Awase, M.K., Atari, A.W., Obayashi, M.K.,

Amesada, M.T., Agi, K.Y., Hepatoprotective Effect of Syringic Acid and Vanillic Acid on

CCl4-. Biological and Pharmaceutical Bulletin, 2010. 33(6): p. 983–987.

80. Kaur, B., Chakraborty, D., Biotechnological and molecular approaches for vanillin

production: a review. Applied biochemistry and biotechnology, 2013. 169(4): p. 1353–1372.

81. Kambourakis, S., Draths, K.M., Frost, J.W., Synthesis of Gallic Acid and Pyrogallol

from Glucose : Replacing Natural Product Isolation with Microbial Catalysis. Journal of

the American Chemical Society, 2000. 122(10): p. 9042–9043.

106

82. Hua, D., Ma, C., Lin, S., Song, L., Deng, Z., Maomy, Z., Biotransformation of

isoeugenol to vanillin by a newly isolated Bacillus pumilus strain : Identification of major

metabolites. Journal of Biotechnology, 2007. 130: p. 463–470.

83. Di Gioia, D., Luziatelli, F., Negroni, A., Ficca, A.G., Fava, F., Ruzzi, M., Metabolic

engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid.

Journal of biotechnology, 2010. 156(4): p. 309–316.

84. Civolani, C., Barghini, P., Roncetti, R., Ruzzi, M., Schiesser, A., Bioconversion of

ferulic acid into vanillic acid by means of a vanillate-negative mutant of Pseudomonas

fluorescens strain BF13. Applied and environmental microbiology, 2000. 66(6): p. 2311–

2317.

85. Abdelkafi, S., Labat, M., Gam, Z.B.A., Lorquin, J., Casalot, L., Sayadi, S., Optimized

conditions for the synthesis of vanillic acid under hypersaline conditions by Halomonas

elongata DSM 2581T resting cells. World Journal of Microbiology and Biotechnology,

2007. 24(5): p. 675–680.

86. Fellows, P., Food processing technology; principles and Practice. second edition.

Cambridge: Woodhead Publishing Limited; 2000. p. 140–168.

87. Grandison, A.S., Lewis, M.J., Separation Processes in the Food and Biotechnology

Industries: Principles and Applications. Woodhead; 1996

88. Ge, Y., Ni, Y., Yan, H., Chen, Y., Cai, T., Optimization of the Supercritical Fluid

Extraction of Natural Vitamin E from Wheat Germ Using Response Surface Methodology.

Journal of food science, 2002. 67(1): p. 239–243.

89. Palma, M., Taylor, L.T., Varela, R.M., Cutler, S.J., Cutler, H.G., Fractional

extraction of compounds from grape seeds by supercritical fluid extraction and analysis for

antimicrobial and agrochemical activities. Journal of agricultural and food chemistry, 1999.

47(12): p. 5044–5048.

90. Farías-Campomanes, A.M., Rostagno, M. A., Meireles, M.A., Production of

polyphenol extracts from grape bagasse using supercritical fluids: Yield, extract

composition and economic evaluation. The Journal of Supercritical Fluids, 2013. 77: p. 70–

78

91. Giergielewicz-Możajska, H., Dąbrowski, Ł., Namieśnik, J., Accelerated Solvent

Extraction (ASE) in the Analysis of Environmental Solid Samples — Some Aspects of Theory

and Practice. Critical Reviews in Analytical Chemistry, 2001. 31(3): p. 149–165.

92. Skalicka-Woźniak, K., Głowniak, K., Quantitative Analysis of Phenolic Acids in

Extracts Obtained from the Fruits of Peucedanum alsaticum L. and Peucedanum cervaria

(L.) Lap. Chromatographia, 2008. 68: p. 85–90.

93. Júnior, D.S., Krug, F.J., Pereira, M.D.G., Korn, M., Currents on Ultrasound‐Assisted

Extraction for Sample Preparation and Spectroscopic Analytes Determination. Applied

Spectroscopy Reviews, 2006. 41(3): p. 305–321.

94. Eskilsson, C.S., Björklund, E., Analytical-scale microwave-assisted extraction.

Journal of Chromatography A, 2000. 902(1): p. 227–250.

95. Li, H., Chen, B., Zhang, Z., Yao, S., Focused microwave-assisted solvent extraction

and HPLC determination of effective constituents in Eucommia ulmodies Oliv. (E.

ulmodies). Talanta, 2004. 63(3): p. 659–665.

96. Teng, H., Lee, W.Y., Choi, Y.H., Optimization of microwave-assisted extraction for

anthocyanins, polyphenols, and antioxidants from raspberry (Rubus Coreanus Miq.) using

response surface methodology. Journal of separation science, 2013. 36(18): p. 3107–3114.

97. Sharma, A., Verma, S.C., Saxena, N., Chadda, N., Singh, N.P., Sinha, A.K.,

Microwave- and ultrasound-assisted extraction of vanillin and its quantification by high-

107

performance liquid chromatography in Vanilla planifolia. Journal of Separation Science,

2006. 29(5): p. 613–619.

98. Dai J., Mumper R.J., Plant phenolics: extraction, analysis and their antioxidant and

anticancer properties. Molecules, 2010. 15(10): P. 7313–7352.

99. Walter, H., Johansson, G., Aqueous Two-Phase Systems. Academic Press, 1994.

100. Zaslavsky, B.Y., Aqueous Two-Phase Partitioning: Physical Chemistry and

Bioanalytical Applications. Taylor and Francis, 1994.

101. Freire, M.G., Cláudio, A.F.M., Araújo, J.M.M., Coutinho, J.P., Marrucho, I.M.,

Canongia Lopes, J.N., Rebelo, L.P.N., Aqueous biphasic systems: a boost brought about by

using ionic liquids. Chemical Society Reviews, 2012. 41(14): p. 4966–4995.

102. Raja, S., Murty, V.R., Thivaharan, V., Rajasekar, V., Ramesh, V., Aqueous Two

Phase Systems for the Recovery of Biomolecules – A Review. Science and Technology, 2012.

1(1): p. 7–16.

103. Reis, I.A.O., Santos, S.B., Santos, L.A., Oliveira, N., Freire, M.G., Pereira, J.F.B.,

Ventura, S.P.M., Coutinho, J.A.P., Soares, C.M.F., Lima, A.S., Increased significance of

food wastes : Selective recovery of added-value compounds. Food Chemistry, 2012. 135(4):

p. 2453–2461.

104. Hasmann, F.A., Santos, C., Gurpilhares, D.B., Roberto, C., Aqueous two-phase

extraction using thermoseparating copolymer : a new system for phenolic compounds

removal from hemicelullosic hydrolysate. Journal of Chemical Technology and

Biotechnology, 2008. 173: p. 167–173

105. Seddon, K.R., Stark, A., Torres, M.J., Influence of chloride, water, and organic

solvents on the physical properties of ionic liquids. Pure and Applied Chemistry, 2000.

72(12): p. 2275–2287.

106. Walden, P., Ueber die Molekulargrösse und elektrische Leitfähigkeit einiger

geschmolzenen Salze. (Molecular weights and electrical conductivity of several fused salts.).

Bull. l´Aacadémie Impériale des Sciences St.-pétersbourg, 1914. 469(1907).

107. Graenacher, C., Cellulose solution. 1934, US Patent 1943176.

108. Hurley, H.F., Wier, J.T.P., Electrodeposition of aluminum. 1948, US Patent

2446331.

109. Fry, S.E., Pienta, N.J., Effects of molten salts on reactions. Nucleophilic aromatic

substitution by halide ions in molten dodecyltributylphosphonium salts. Journal of the

American Chemical Society, 1985. 107(22): p. 6399–6400.

110. Boon, J.A., Levisky, J.A., Pflug, J.L., Wilkes, J.S., Friedel-Crafts reactions in

ambient-temperature molten salts. The Journal of Organic Chemistry, 1986. 51(4): p. 480–

483.

111. Wilkes, J.S., Zaworotko, M.J., Air and water stable 1-ethyl-3-methylimidazolium

based ionic liquids. Journal of the Chemical Society, 1992. (13): p. 965–967.

112. Ranke, J., Stolte, S., Störmann, R., Arning, J., Jastorff, B., Design of Sustainable

Chemical Products. The Example of Ionic Liquids. Chemical Reviews, 2007. 107(6): p.

2183–2206.

113. Wasserscheid, P., Welton, T., Ionic liquids in synthesis. Wiley, 2007. p. 689–704.

114. Earle, M.J., Esperanca, J.M.S.S., Gilea, M.A., Canongia Lopes, J.N., Rebelo,

L.P.N., Magee, J.W., Seddon, K.R., Widegren, J.A. The distillation and volatility of ionic

liquids. Nature, 2006. 439(7078): p. 831–834.

115. Rogers, R.D., Seddon, K.R., Ionic Liquids--Solvents of the Future? Science, 2003.

302(5646): p. 792–793.

108

116. Pandey, S., Analytical applications of room-temperature ionic liquids: a review of

recent efforts. Analytica Chimica Acta, 2006. 556(1): p. 38–45.

117. Gutowski, K.E., Broker, G., Willauer, H.D., Huddleston, J.G., Swatloski, R.P.,

Holbrey, J.D., Controlling the aqueous miscibility of ionic liquids: aqueous biphasic systems

of water-miscible ionic liquids and water-structuring salts for recycle, metathesis, and

separations. Journal of the American Chemical Society, 2003. 125(22): p. 6632–6633.

118. Domínguez, C.M., Munoz, M., Quintanilla, A., Pedro, Z.M., Ventura, S.P.M.,

Coutinho, J.A P., Casas, J.A., Rodriguez, J.J., Degradation of imidazolium-based ionic

liquids in aqueous solution by Fenton oxidation. Journal of Chemical Technology &

Biotechnology, 2014. 89(8): p. 1197-1202.

119. Khan, I., Kurnia, K.A., Sintra, T.E., Saraiva, J.A., Pinho, S.P., Coutinho, J.A.P.,

Assessing the activity coefficients of water in cholinium-based ionic liquids: Experimental

measurements and COSMO-RS modeling. Fluid Phase Equilibria, 2014. 361: p. 16–22.

120. Pereira, J.F.B., Vicente, F., Santos-Ebinuma, V.C., Araújo, J.M., Pessoa, A., Freire,

M.G., Coutinho, J.A.P., Extraction of tetracycline from fermentation broth using aqueous

two-phase systems composed of polyethylene glycol and cholinium-based salts. Process

Biochemistry, 2013. 48(4): p. 716–722.

121. Pereira, J.F.B., Kurnia, K.A., Cojocaru, O.A., Gurau, G., Rebelo, L.P.N., Rogers,

R.D., Freire, M.G., Coutinho, J.A.P., Molecular interactions in aqueous biphasic systems

composed of polyethylene glycol and crystalline vs. liquid cholinium-based salts. Physical

Chemistry Chemical Physics : PCCP, 2014. 16(12): p. 5723–5731.

122. Shahriari, S., Tomé, L.C., Araújo, J.M.M., Rebelo, L.P.N., Coutinho, J.A.P.,

Marrucho, I.M., Freire, M.G., Aqueous biphasic systems: a benign route using cholinium-

based ionic liquids. The Royal Society of Chemistry, 2013. 3(6): p. 1835-1843.

123. Cláudio, A.F.M., Ferreira, A.M., Freire, C.S.R., Silvestre, A.J.D., Freire, M.G.,

Coutinho, J.A.P., Optimization of the gallic acid extraction using ionic-liquid-based

aqueous two-phase systems. Separation and Purification Technology, 2012. 97: P. 142–149.

124. Cláudio, A.F.M., Freire, M.G., Freire, C.S.R., Silvestre, A.J.D., Coutinho, J.A.P.,

Extraction of vanillin using ionic-liquid-based aqueous two-phase systems. Separation and

Purification Technology, 2010. 75: p. 39–47.

125. Reis, I.A.O., Santos, S.B., Pereira, F.D.S., Sobral, C.R.S., Freire, M.G., Freitas,

L.S., Soares, C.M.F., Lima, A.S., Extraction and Recovery of Rutin from Acerola Waste

using Alcohol-Salt-Based Aqueous Two-Phase Systems. Separation Science and

Technology, 2014. 49(5): p. 656–663.

126. Liu, Y., Wu, Z., Zhang, Y., Yuan, H., Partitioning of biomolecules in aqueous two-

phase systems of polyethylene glycol and nonionic surfactant. Biochemical Engineering

Journal, 2012. 69: p. 93–99.

127. Pereira, J.F.B., Rebelo, L.P.N., Rogers, R.D., Coutinho, J.A.P., Freire, M,G.,

Combining ionic liquids and polyethylene glycols to boost the hydrophobic-hydrophilic

range of aqueous biphasic systems. Physical chemistry chemical physics, 2013. 15(45):p.

19580–19583.

128. Lopes, N.C., Rebelo, L.P.N., Visak, Z.P., Ionic Liquids in Polyethylene Glycol

Aqueous Solutions : Salting-in and Salting-out Effects. Chemical Monthly, 2007. 138: p.

1153–1157.

129. Zafarani-Moattar, M.T., Hamzehzadeh, S., Nasiri, S., A new aqueous biphasic

system containing polypropylene glycol and a water-miscible ionic liquid. Biotechnology

Progress, 2012. 28: p. 146–156.

109

130. Wu, C.Z., Wang, J.J., Pei, Y.C., Wang, H.Y., Li, Z.Y., Salting-Out Effect of Ionic

Liquids on Polypropylene glycol) (PPG): Formation of PPG + Ionic Liquid Aqueous Two-

Phase Systems. Journal of Chemical and Engineering Data, 2010. 55: p. 5004–5008.

131. Freire, M.G., Pereira, J.F.B., Francisco, M., Rodríguez, H., Rebelo, L.P.N., Rogers,

R.D., Coutinho, J.A.P., Insight into the interactions that control the phase behaviour of new

aqueous biphasic systems composed of polyethylene glycol polymers and ionic liquids.

Chemistry, 2012. 18(6): p. 1831–1839.

132. Almeida, M.R., Passos, H., Pereira, M.M., Lima, Á.S., Coutinho, J.A.P., Freire,

M.G., Ionic liquids as additives to enhance the extraction of antioxidants in aqueous two-

phase systems. Separation and Purification Technology. Elsevier B.V., 2014. 128: p. 1–10.

133. Walter R., Process of producing n-vinyl compounds. 1939. US2153993 A.

134. Buehler, V., Kollidon. Polyvinylpyrrolidone excipients for the pharmaceutical

industry, 2008. 9th Ed: p. 11-134.

135. Haaf, F., Sanner, A., Straub, F., Polymers of N-Vinylpyrrolidone: Synthesis,

Characterization and Uses. Polymer Journal, 1985. 17(1): p. 143–152.

136. Folttmann, B.H., Quadir, A., Polyvinylpyrrolidone (PVP) - One of the Most Widely

Used Excipients in Pharmaceuticals : An Overview. Drug Delivery Technology, 2008. 8(6):

p. 22-27.

137. Forster, A., Hempenstall, J., Rades, T., Characterization of glass solutions of poorly

water-soluble drugs produced by melt extrusion with hydrophilic amorphous polymers. The

Journal of pharmacy and pharmacology, 2001. 53(3): p. 303–315.

138. U.S. Department of Health and Human Services, FDA/Center for Drug Evaluation

and Research. Inactive Ingredients in FDA Approved Drugs. Available from: www.fda.gov.

139. Rogers, R.D., Zhang, J., Effects of increasing polymer hydrophobicity on

distribution ratios of TcO4- in polyethylene/poly(propylene glycol)-based aqueous biphasic

systems. Journal of Chromatography B: Biomedical Applications, 1996. 680(17): p. 231–

236.

140. Baxter, S.M., Sperry, P.R., Fu, Z., Partitioning of Polymer and Inorganic Colloids

in Two-Phase Aqueous Polymer Systems. Langmuir, 1997. 13(15): p. 3948–3952.

141. Zaslavsky, B.Y., Bagirovl, T.O., Borovskaya, A.A., Gasanoval, G.Z., Gulaeva,

N.D., Levin, V.Y., et al.,. Aqueous biphasic systems formed by nonionic polymers I . Effects

of inorganic salts on phase separation. Colloid & Polymer Science, 1986. 264: p. 1066–

1071.

142. Pernak, J., Syguda, A., Mirska, I., Pernak, A., Nawrot, J., Pra̧dzyńska, A., Griffin,

S.T., Rogers, R.D., Choline-Derivative-Based Ionic Liquids. Chemistry – A European

Journal, 2007. 13(24): p. 6817–6827.

143. Muhammad, N., Hossain, M.I., Man, Z., El-Harbawi, M., Bustam, M.A., Noaman,

Y.A., Alitheen, N.B.M., Ng, M.K., Hefter, G., Yin, C.Y., Synthesis and Physical Properties

of Choline Carboxylate Ionic Liquids. Journal of Chemical & Engineering Data, 2012. 57(8):

p. 2191–2196.

144. Merchuk, J.C., Andrews, B.A., Asenjo, J.A., Aqueous two-phase systems for protein

separation Studies on phase inversion. Journal of Chromatography B, 1998. 711(1-2): p.

285–293.

145. Louros, C.L.S., Cláudio, A.F.M., Neves, C.M.S.S., Freire, M.G., Marrucho, I.M.,

Pauly, J., Coutinho, J.A.P, Extraction of biomolecules using phosphonium-based ionic

liquids + K3PO4 aqueous biphasic systems. International Journal of Molecular Sciences,

2010. 11(4): p. 1777–1791.

110

146. Passos, H., Trindade, M.P., Vaz, T.S.M., Costa, L.P., Freire, M.G., Coutinho, J.A.P.,

The impact of self-aggregation on the extraction of biomolecules in ionic-liquid-based

aqueous two-phase systems. Separation and Purification Technology, 2013. 108: p. 174–

180.

147. Carvalho, S.F., Aqueous Biphasic Systems Composed of Ionic Liquids and

Polysaccharides. Master Thesis, University of Aveiro, 2013.

148. Ferreira, A.M., Coutinho, J.A.P., Fernandes, A.M., Freire, M.G., Complete removal

of textile dyes from aqueous media using ionic-liquid-based aqueous two-phase systems.

Separation and Purification Technology. Elsevier B.V., 2014. 128: p. 58–66.

149. Baker, M.I., Walsh, S.P., Schwartz, Z., Boyan, B.D., A review of polyvinyl alcohol

and its uses in cartilage and orthopedic applications. Journal of Biomedical Materials

Research Part B, Applied Biomaterials, 2012. 100(5): p. 1451–1457.

150. Kadajji, V.G., Betageri, G.V., Water Soluble Polymers for Pharmaceutical

Applications. Polymers, 2011, 3(4): p. 1972–2009.

151. Hassan, C.M., Peppas, N.A., Structure and Applications of Poly(vinyl alcohol)

Hydrogels Produced by Conventional Crosslinking or by Freezing / Thawing Methods.

Advances in Polymer Science, 2000. 153: p. 38-62.

152. Patachia, S., Friedrich, C., Florea, C., Croitoru, C., Study of the PVA hydrogel

behaviour in 1-butyl-3-methylimidazolium tetrafluoroborate ionic liquid. Express Polymer

Letters, 2011. 5(2): p. 197–207.

153. Domínguez-Pérez, M., Tomé, L.I.N., Freire, M.G., Marrucho, I.M., Cabeza, O.,

Coutinho, J.A.P., (Extraction of biomolecules using) aqueous biphasic systems formed by

ionic liquids and aminoacids. Separation and Purification Technology, 2010. 72(1): p. 85–

91.

111

6. Publications

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.

115

Appendix A

Calibration curves

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

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



[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



[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



[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



[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.


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.


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

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

-

- -


-

-


-

-


-

- -

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


-


-


-

-

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


-


-

-

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

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