extraction of bioactive compounds with ionic liquid
TRANSCRIPT
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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
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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
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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
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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
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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 %
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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
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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
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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
4. Results and discussion 4.1. IL + H2O + C6H5K3O7 ternary systems
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
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ATPS composed of IL + Potassium citrate + H2O
4. Results and discussion 4.1. IL + H2O + C6H5K3O7 ternary systems
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
)
[C6H5K3O7] (mol∙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
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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
)
[C6H5K3O7] (mol∙kg-1)
ATPS composed of IL + Potassium citrate + H2O
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
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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
ATPS composed of IL + Potassium citrate + H2O
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
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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.
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4. Results and discussion 4.2. Self-aggregation in IL-based ATPS
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
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Imidazolium-based IL + C6H5K3O7/C6H8O7 + alkaloids
n ≥ 7 IL ability for the micelles formation KAlk 14
4. Results and discussion 4.2. Self-aggregation in IL-based ATPS
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
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4. Results and discussion 4.2. Self-aggregation in IL-based ATPS
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
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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
4. Results and discussion 4.2. Self-aggregation in IL-based ATPS
Imidazolium-based IL + C6H5K3O7 + alkaloids
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-
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0
20
40
60
80
100
0 2 4 6 8 10 12 14
Co
nce
ntr
atio
n (
wt
%)
pH 17
4. Results and discussion 4.2. Self-aggregation in IL-based ATPS
Imidazolium-based IL + C6H5K3O7 + alkaloids
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
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Micelles formation repulsive forces KNic
0
4
8
12
16
20
24 KAlk
pH 7 pH 9
18
4. Results and discussion 4.2. Self-aggregation in IL-based ATPS
Imidazolium-based IL + C6H5K3O7 + alkaloids Nicotine
H+
repulsive forces
Nicotine is the most hydrophobic compound studied stay inside of micelles
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4. Results and discussion 4.2. Self-aggregation in IL-based ATPS
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
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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
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4. Results and discussion 4.3. Extraction of bisphenol A (BPA)
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
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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
4. Results and discussion 4.3. Extraction of bisphenol A (BPA)
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).
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95
96
97
98
99
100
EEBPA%
Aqueous Phase Artificial human urine
Cl-
N+
N
23
4. Results and discussion 4.3. Extraction of bisphenol A (BPA)
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
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95
96
97
98
99
100
22 wt % 25 wt % 26 wt % 28 wt % 31 wt %
EEBPA%
4. Results and discussion 4.3. Extraction of bisphenol A (BPA)
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
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95
96
97
98
99
100
EEBPA%
[C2mim]Cl [N1112OH]Cl
25
4. Results and discussion 4.3. Extraction of bisphenol A (BPA)
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.
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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
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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);