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Extraction of bioactive compounds with ionic liquid

aqueous solutions

Helena Passos

universidade de aveiro

dqua departamento de química

Dissertação/Projeto 5ºAno

Mestrado Integrado em Engenharia Química

20 de Julho de 2012

Orientadores

Prof. Dr. João Coutinho

Dr.ª Mara Freire


Summary

2

1. Introduction

1.1. Ionic liquids (ILs)

1.2. Aqueous two phase systems (ATPS)

2. Objectives and scopes

3. Experimental procedure

4. Results and discussion

4.1. IL + H2O + C6H5K3O7 ternary systems

4.2. Self-aggregation in IL-based ATPS

4.3. Extraction of bisphenol A (BPA)

5. Conclusions

6. Future work


Designer solvents

Volatile organic compounds substitutes

negligible vapour pressure and flammability;

high thermal and chemical stability;

high solvation ability for several compounds;

improved selectivity and easy recycling;

tunable properties.

1. Introduction 1.1. Ionic liquids (ILs)

3

ILs are composed of

large organic cations

organic or inorganic anions

N+

R1

R2R3

R4

N+

R

N+

N

R2

R1

Imidazolium Pyridinium

Ammonium

P

O

OO

O

N

C C

N N

Cl-

Chloride

Dimethylphosphate

Dicyanamide

exhibit unique properties


4

1. Introduction 1.2. Aqueous two phase systems (ATPS)

ATPS composed of IL + Salt + H2O.

Liquid-Liquid Extraction

ATPS

two aqueous-rich phases

polymer

salt

salt

+

+

+

polymer

polymer

salt

Additional advantages

low viscosity;

quick phase separation;

high and tailored extraction efficiency.

IL-based ATPS


2. Objectives and scopes

To demonstrated the potential of IL-based ATPS for the

extraction of Bisphenol A (BPA).

To study the ability of novel ATPS for alkaloids extraction;

To study the effect of the ILs self-aggregation and

subsequent impact on the partition of different biomolecules;

5

To find more benign ATPS by substitution of the

high charge density salts usually employed;

0

10

20

30

40

50

60

70

0 10 20 30

[IL]

/ w

t %

[C6H5K3O7] / wt %


3. Experimental procedure

6

A

B

Cloud point titration method. ATPS composed of IL + Salt + H2O.

IL-rich phase

Salt-rich phase

Determination of phase diagrams

Cloud point titration method

Extraction of bioactive compounds

1. ATPS preparation: IL + Salt + Biomolecules

2. Phases separation

3. UV-spectroscopy

quantification

4. Determination of:

%100%

Salt

X

IL

X

IL

XX

mm

mEE

Salt

ILX

X

XK

Partition Coefficient

Extraction Efficiency


replaced by biodegradable and biocompatible organic salts

4. Results and discussion 4.1. IL + H2O + C6H5K3O7 ternary systems

IL-based ATPS are commonly composed by

inorganic salts

environmental concern

7


ATPS composed of IL + Potassium citrate + H2O

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0

[IL]

(m

ol∙

kg-1

)

[C6H5K3O7] (mol∙kg-1)

0.0

1.0

2.0

3.0

4.0

0.0 0.4 0.8 1.2 1.6


1. Cation core effect

Cl-

- [C4mpy]Cl

[P4444]Cl [N4444]Cl

[C4mim]Cl

[C4mpip]Cl

+ [C4mpyr]Cl

N +

N Cl-

Cl-

Cl-

Cl- Cl-

[P4444]+ > [N4444]

+ >> [C4mpy]+ ≈ [C4mpip]+ > [C4mpyr ]+ > [C4mim]+ 8




0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0.0 0.5 1.0 1.5 2.0 2.5

[IL]

(m

ol∙

kg-1

)


2. Anion effect

N +

N

[CH3CO2]- < [PO4(CH3)2]

- < [CH3SO3]- < Cl- << Br- < [CF3CO2]

- << [N(CN)2]- <

[SCN]- < [CF3SO3]-

- [C4mim][CF3SO3]

[C4mim]Br

[C4mim][CF3CO2]

[C4mim]Cl

[C4mim][SCN]

+ [C4mim][N(CN)2]

[C4mim][CH3SO3]

[C4mim][CH3CO2]

[C4mim][PO4(CH3)2]

[C4mim]+ cation

9

hyd

rog

en

bo

nd

basic

ity


10

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0.0 0.5 1.0 1.5 2.0 2.5

[IL]

(m

ol∙

kg-1

)



4. Results and discussion 4.1. ILs + H2O + C6H5K3O7 ternary systems

3. Cation alkyl side chain length effect

[C4mim]- < [C6mim]- ≈ [C7mim]- ≈ [C8mim]- ≈ [C10mim]-

[C6mim]Cl

[C7mim]Cl

[C8mim]Cl

[C10mim]Cl

[C4mim]Cl

[Cnmim]Cl

N+

N R1

Cl- n hydrophobicity solubility

ability for ATPS formation


0

10

20

30

40

50

60

70

80

0.0 0.5 1.0 1.5 2.0 2.5

[IL]

(w

t %

)

[Salt] (mol∙kg-1)

11


4. Results and discussion 4.1. ILs + H2O + C6H5K3O7 ternary systems

4. pH effect

pH 9 pH 7

[C4mim]Cl

pH 9 pH 7

[C10mim]Cl

N +

N Cl-

N+

NCl-

pH effect is negligible


12

4. Results and discussion 4.2. Self-aggregation in IL-based ATPS

[Cnmim]+ are structurally similar to ionic surfactants

ILs micelle formation in aqueous solution[1]

[1] C. Jungnickel et al., Colloids and Surfaces A: Physicochem. Eng. Aspects 316 (2008) 278–284.

N+

N

N+

N

N+

N

N+

N

N+

N

N+

N

N+

N

N+

N

N+

NCl-

Cl- Br-

N+

NS-N

Cl- Cl-

Cl-

Cl-

Cl-

i ii iii

iv v vi

vii viii ix

x

xvii xviii

xiv xv

xvi

N+

NCl-

N+

N

N+

NCl- Cl-

xi

xii xiii

Hydrophilic head

Hydrophobic chain

[C10mim]- molecular structure.


13


Micelle-mediated extraction can be used to increase or decrease the

extraction efficiencies of a given molecule.

[3] A.F.M. Cláudio et al., Separation and Purification Technology 75 (2010) 39–47.

[2] M.G. Freire et al., Green Chem. 12 (2010) 1715–1718.

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

High-performance extraction of alkaloids using aqueous two-phase systems with ionic liquids


Imidazolium-based IL + C6H5K3O7/C6H8O7 + alkaloids

n ≥ 7 IL ability for the micelles formation KAlk 14


Caffeine

Theophylline

Theobromine

0

2

4

6

8

10

12

14 KAlk

Caffeine Theophylline Theobromine

Maximum of extraction [C6mim]Cl

pH ≈ 7

Alkaloids are predominantly in a non-charged form


15


Imidazolium-based IL + C6H5K3O7 + alkaloids

- pH ≈ 9

Theophylline is predominantly in a negative charged form

0

20

40

60

80

100

0 2 4 6 8 10 12 14

Co

nce

ntr

atio

n (

wt

%)

pH

Theophylline


0

3

6

9

12

15

18

KAlk

pH 7 pH 9

-

Theophylline

KAlk for charged molecule > KAlk for neutral molecule n interaction between opposite charges n ≥ 8 micelles formation KAlk

16



T-

T-

T-

T-

T-

T-

N +N

N +N

N+

N

N+

N

N +N

N+

N

Cl-

Cl-

Cl-

Cl-

Cl- Cl-

N+

N

+

N+ N+

N+

N

+

N+N +

N+

N

+

T-

Cl-

T-

Cl- T-

Cl-

T-

Cl-

T-

Cl-

T-

T-

T-

T-

T- T-

T- T-

T-

T-

T-

T-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-


0

20

40

60

80

100

0 2 4 6 8 10 12 14

Co

nce

ntr

atio

n (

wt

%)

pH 17



pH ≈ 9 Nicotine is preferentially in a neutral form (58 wt % of neutral molecule and 42 wt % of positively charged

molecule)

Nicotine

H+

pH ≈ 7 Nicotine is predominantly in a positive charged form


Micelles formation repulsive forces KNic

0

4

8

12

16

20

24 KAlk

pH 7 pH 9

18


Imidazolium-based IL + C6H5K3O7 + alkaloids Nicotine

H+

repulsive forces

Nicotine is the most hydrophobic compound studied stay inside of micelles


19


Imidazolium-based IL + C6H5K3O7 Microscopy aplication

ATPS composed of IL + Salt + H2O.

Microscope image of IL-rich phase of ATPS composed of [C8mim]Cl + C6H5K3O7 + H2O.

Confirmed the presence of micelles in systems composed of [C7mim]Cl, [C8mim]Cl and [C10mim]Cl


Applications of BPA

- Plastic industry as an intermediate in

the production of epoxy resins and

polycarbonate plastics;

- In the manufacture of thermal paper.

However...

BPA is an endocrine disruptor

• Heart disease;

• Obesity;

• Breast and prostate cancer;

• Neurobehavioral problems;

• Infertility;

• etc…

BPA is now an ubiquitous component in the atmosphere

Production:3 million tons/year

Release into the atmosphere: 100 tons/year

4. Results and discussion 4.3. Extraction of bisphenol A (BPA)

BPA

20


21


ATPS composed of

IL + K3PO4 + H2O

IL-based systems formed by the addition

of K3PO4 provide high extraction

efficiencies due to the presence of the

strong salting-out salt


0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0

[IL]

(m

ol∙

kg-1

)

[K3PO4] (mol∙kg-1)

0.0

0.5

1.0

1.5

2.0

0.5 1.0 1.5

Cl-

N+

N

22


ATPS composed of IL + K3PO4 + H2O N +

N

N +

N Cl-

Cl- Cl-

Cl-

N+

NCl-

Cl-

Cl-

[amim]Cl[4]

[P4444]Cl + [N4444]Cl

[C2mim]Cl[4]

[C4mpyr]Cl

[C4mim]Cl[4]

[C6mim]Cl[4]

- [N1112OH]Cl[5]

[P4444]+ > [N4444]

+ >> [C6mim]+ > [C4mpyr]+ > [C4mim]+ > [amim]+ > [C2mim]+ >> [N1112OH]+

[4] Neves, C. M. S. S., et al, J. Phys. Chem. B 2009, 113, 5194–5199; [5] Louros, C.L.S., MSc thesis, “Extraction of Biomolecules with Aqueous Two Phases Systems”, University of Aveiro, Aveiro, Portugal (2009).


95

96

97

98

99

100

EEBPA%

Aqueous Phase Artificial human urine

Cl-

N+

N

23


ATPS composed of IL + K3PO4 + H2O N +

N

N +

N Cl-

Cl- Cl-

Cl-

N+

NCl-

Cl-

Cl-

[amim]Cl[4]

[P4444]Cl + [N4444]Cl

[C2mim]Cl[4]

[C4mpyr]Cl

[C4mim]Cl[4]

[C6mim]Cl[4]

- [N1112OH]Cl[5]

[N1112OH]Cl Maximun of EEBPA% alkyl side chain length EEBPA%

[4] Neves, C. M. S. S., et al, J. Phys. Chem. B 2009, 113, 5194–5199; [5] Louros, C.L.S., MSc thesis, “Extraction of Biomolecules with Aqueous Two Phases Systems”, University of Aveiro, Aveiro, Portugal (2009).

The presence of a more complex matrix, favors

the partitioning of BPA for the IL-rich phase


95

96

97

98

99

100

22 wt % 25 wt % 26 wt % 28 wt % 31 wt %

EEBPA%


0

10

20

30

40

0 10 20 30 40

[IL

] (w

t %

)

[K3PO4] (wt %)

Optimization of amount of IL without

losing the high extraction efficiencies

of BPA

24

TLL [IL]

[C2mim]Cl [N1112OH]Cl

Minimum concentrations:

15 wt % K3PO4 + 25 wt % [C2mim]Cl

22 wt % K3PO4 + 28 wt % [N1112OH]Cl


95

96

97

98

99

100

EEBPA%

[C2mim]Cl [N1112OH]Cl

25


0

10

20

30

40

0 10 20 30 40

[IL

] (w

t %

)

[K3PO4] (wt % )

Maximum concentration of BPA achievable

IL IL IL

Salt Salt

Salt

VIL [BPA] IL-rich phase

The concentration of BPA can be increased at least up to 100-fold.


The organic salt tri-potassium citrate showed to be a good option for the

substitution of commonly used inorganic salts, in ATPS formation;

5. Conclusions

26

For the first time:

The application of IL-based ATPS shows to be an improved technique for

concentrating the levels of BPA from biological fluids;

it was addressed the effect of micelles formation and their impact on the

extraction of (bio)molecules (both charged and non-charged);

For all investigated systems, extraction efficiencies of BPA are higher than

98.5 %.

Significant impact in extraction processes


6. Future work

27

To understand better the mechanisms underlying to the micelle formation in

these systems and their effect in the extraction process;

To study novel ATPS composed of IL and other organic salts and their potential

in the extraction of different types of compounds;

Extraction of BPA with IL-based ATPS:

To work with real body fluid samples, aiming at providing a general

overview of the levels of BPA in the Portuguese population and its relation

with several types of diseases.

To support the finding of the micelle-mediated extraction – application of

transmission electron microscopy (TEM);


Acknowledgements

Path and Mini-Path Thank you for your attention!

Prof. João Coutinho

Dr.ᵃ Mara Freire

Ana Filipa Cláudio