Separation and Purication Technology xxx (2012) xxxxxx

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Separation and Purication Technology

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Ionic liquid-based aqueous two-phase extraction within a microchannel system

Uro Novak, Andrej Pohar, Igor Plazl, Polona Znidaric-Plazl
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Akerceva 5, 1000 Ljubljana, Slovenia

a r t i c l e

i n f o

Article history:
Available online xxxx
Keywords:
Aqueous two-phase system
Microuidics
Ionic liquid
Protein extraction

a b s t r a c t
Ionic liquid-based aqueous two-phase systems (IL-ATPSs) are great candidates for the replacement of volatile organic solvents in liquidliquid extractions. Besides, microuidic separation techniques are a promising alternative to conventional systems since they provide continuous operation, better performance
and easier capacity increase by a numbering-up approach. Herein, the advantages of IL-ATPSs and microuidic continuous separation were combined within a microuidic device with parallel ow allowing for
the separation of the two phases at the exit of the microchannel. An aqueous solution of [C4mim] [BF4]
and D-fructose was used as an IL-ATPS and compared with a conventional ATPS consisted of polyethylene
glycol/salt solution. Based on the evaluated partitioning coefcient of a model substance, namely bovine
serum albumin, the ionic liquid concentration and pH value of the IL-ATPS have been selected for the use
within a pressure-driven microchannel system with y-shaped inlet and outlet. Furthermore, a threedimensional model considering convection in the ow direction and diffusion in all spatial directions
at steady-state conditions was developed, validated by experimental results and used to assess the feasibility of micro-scale parallel ow extraction in a wide range of ow rates. The chosen IL-ATPS was
shown to be much more efcient media for continuous microuidic extraction as compared to conventional ATPS, primarily due to much lower dynamic viscosity of IL-rich phase related to PEG-rich phase.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Aqueous two-phase systems (ATPSs) offer an attractive alternative to conventional extraction methods for the separation of
biomolecules [14]. Due to many advantages such as a biocompatible environment, low energy consumption, relative reliability in
scale up and short process time, they have been recognized as an
economical and efcient downstream processing possibility [5].
Conventional ATPSs are formed by mixing two polymers (e.g. polyethylene glycol, dextran, etc.) or a polymer and an inorganic salt
(e.g. phosphate, sulfate, etc.) with water.
In 2003, Rogers et al. reported that also some hydrophilic ionic
liquids (ILs) are able to form IL-ATPSs when mixed with aqueous
solutions of inorganic salts [6]. Since the toxicity of ILs, which recently raised doubts about the generally accepted view of ILs as
green solvents, is directly related with their hydrophobicity, they
are less toxic than their more hydrophobic counterparts [7]. Even
more environmentally benign IL-ATPSs consisting of hydrophilic
ILs and sugars were lately described, which are suggested to be
more appropriate for recycling ILs than those of ILs and inorganic
salts [811]. lL-ATPSs were found particularly suitable for the
extraction of proteins, where the selectivity and even stabilization

Corresponding author. Tel.: +386 1 2419 533.

E-mail address: polona.znidarsic@fkkt.uni-lj.si (P. Znidaric-Plazl).

of biomolecules might be achieved by the proper IL selection [12

14].
Microuidic devices and microreactor technology are gaining
increased attention over the past 15 years. The main idea behind
this new approach is that within microstructured devices the process control is radically improved due to the increased mass and
heat transfer, which also lead to process intensication. Due to
the exible manufacturing processes of microuidic structures,
the reactors can be designed to t the required chemistry in contrast to the conventional procedure, where the chemistry is
pressed into a given plant [15].
The research on microchannel separation systems is scarcer as
compared to reactions within microreactors, and is mainly devoted
to analytical applications. Studies on separations within microuidic devices revealed higher selectivity and yield together with
the reduced consumption of resources and solvents, primarily
due to the very large surface-to-volume ratio and small dimensions
of the separation units. Therefore microuidic approach has a variety of current and potential applications, especially regarding the
possibility for parallel operations [1621].
The use of ATPS within a microuidic device for protein purication was recently reported by Meagher et al. who demonstrated
continuous microuidic version of a single-stage PEG/salt aqueous
two-phase extraction process using genetically tagged proteins to
drive partitioning into the PEG-rich phase [22]. The purication
of a membrane protein from crude cell extract was achieved also

1383-5866/$ - see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.seppur.2012.01.033

Please cite this article in press as: U. Novak et al., Ionic liquid-based aqueous two-phase extraction within a microchannel system, Separ. Purif. Technol.
(2012), doi:10.1016/j.seppur.2012.01.033

U. Novak et al. / Separation and Purication Technology xxx (2012) xxxxxx

by a continuous microuidic extraction using ATPS consisted of

PEG/detergent, resulting in signicantly increased extraction speed
and efciency as compared to the conventional discontinuous agitation method [23]. Electrophoretic partitioning of proteins by the
diffusive transport of proteins across a uid phase boundary was
also achieved using ATPS [24]. Recently, the microuidic separation system using ATPS and IL-based ATPS was used for the effective extraction and purication of bacteriorhodopsin from the
purple membrane of Halobacterium salinarium [25].
In this work, an experimental and theoretical study of the continuous parallel ow microuidic IL-based aqueous two-phase
extraction within a microuidic device with phase separation at
the exit of the microchannel was performed and compared to conventional ATPS based on PEG/phosphate. The hydrophilic IL
[C4mim] [BF4] and D-fructose were employed for the formation of
the IL-ATPS and bovine serum albumin (BSA) was used as a model
protein. The effects of the IL concentration and pH value on the
partitioning coefcient for BSA have been investigated in batch
experiments. Furthermore, a three-dimensional model considering
convection in the ow direction and diffusion in all spatial directions at steady-state conditions was developed, comprising the
uid ow pattern of two parallel phases at steady-state conditions
in order to analyze experimental data and to forecast microextraction unit performance.
2. Theoretical background
2.1. Velocity prole in a microchannel
Under microuidic conditions it is typical to have a laminar
ow, which enables even two miscible uids to ow next to each
other without turbulent mixing and without physical separation
with a membrane [26]. In order to simulate BSA extraction in a
microchannel system, the velocity prole was rst set up. As
shown in Fig. 1, the microchannel was fed by two inows: f1 as a
ow of phase 1 (phosphate-rich or D-fructose-rich phase of ATPS
or IL-ATPS, respectively) with dissolved BSA and f2 as a ow of
phase 2 (PEG or IL-rich phase of ATPS or IL-ATPS, respectively).
Since the phases had different viscosities, ow rates had to be
adjusted so that the two phases occupied the same fraction of
the channel. The assumption of the parabolic velocity prole,

developed only in the smallest x-dimension, and therefore the uniform velocity prole in the z-direction was not reliable due to the
small (below 10) width/height ratio of the microchannels used in
our experiments. Furthermore, compressibility and gravitational
force were neglected and continuity and momentum equations
for a fully developed Poiseuille type ow were solved [18,27].
2.2. A model for BSA extraction in a microchannel
For the description and prediction of microextractor performance, a 3D model was developed considering convection in the
ow direction and diffusion in all directions. Due to the known
interfacial area in the middle of the microchannel, mass transfer
of protein molecules from phase 1 into phase 2 could be described
using partial differential equations for steady-state conditions in
the single pass microchannel extractor system:

@c
@ 2 c1 @ 2 c 1 @ 2 c 1
v 1 x; y 1 D1
2 2
@z
@x2
@y
@z

v 2 x; y

@c2
@ 2 c2 @ 2 c 2 @ 2 c 2
2 2
D2
@z
@x2
@y
@z

!
1
!
2

with the associated boundary conditions for both phases

C 1 x; y; 0 c1;in

06x6H

0 6 y 6 W2

C 2 x; y; 0 c2;in

06x6H

W
2

@c1 x;y;L
@z

06x6H

0 6 y 6 W2

@c2 x;y;L
@z

06x6H

W
2

06y6

W
2

0<z<L

W
2

0<z<L

@c1 0;y;z
@x
@c1 H;y;z
@x

<y6W

<y6W

06y6

@c2 0;y;z
@x

W
2

<y6W

0<z<L

@c2 H;y;z
@x

W
2

<y6W

0<z<L

@c1 x;0;z
@y

0<x<H

0<z<L

0<x<H
 W 
; z K p c2 x; 2 ; z 0 < x < H

0<z<L

@c2 x;W;z
@y

c1 x; W2
D1

@c1 x;W
;z
2
@y

D2

@c2 x;W
;z
2
@y

0<x<H

0<z<L
0<z<L

Fig. 1. A scheme of (a) a whole microuidic chip and (b) the section of the main microchannel.

Please cite this article in press as: U. Novak et al., Ionic liquid-based aqueous two-phase extraction within a microchannel system, Separ. Purif. Technol.
(2012), doi:10.1016/j.seppur.2012.01.033

U. Novak et al. / Separation and Purication Technology xxx (2012) xxxxxx

where v1 and v2 are the linear velocities of phase 1 (phosphate-rich

or D-fructose-rich phase of ATPS or IL-ATPS, respectively) and phase
2 (PEG-rich phase of ATPS or IL-rich phase of IL-ATPS) inside the
microchannel system. D1 and D2 are molecular diffusion coefcients
for BSA and c1 and c2 are concentrations of BSA in phase 1 and phase
2, respectively, while Kp is the partitioning coefcient for BSA in
ATPS or IL-ATPS. Considering the laminar ow and the equality of
the velocities of both phases at the interface, v1(W/2,z) = v2(W/
2,z), the transfer of protein across the liquidliquid interface was
described by the diffusional mass transport. The equilibrium relation between the concentration of BSA in both phases at the interface c1(x,W/2,z) = Kpc2(x,W/2,z) was taken as boundary condition at
the phase 1 side interface, while the continuity of ux on both sides
of the interface was considered as a boundary condition at the
phase 2 interface [Eq. (3)].
2.3. Numerical analysis
Finite differences were used to replace the partial derivatives in
the presented model equations. The discretization was done by the
nite differences on a 3D Cartesian grid which demands the implicit approach of solution. The formulation of the mathematical
model in the microchannel can only be solved by rigorous numerical procedures, especially for the complex geometry of the domain. In order to solve the complex system of model equations,
Matlab code was developed which enabled fast converging to the
solution. The numerical experiments were analyzed and examined
for accuracy, stability, and theoretical consistency. Static equidistant nite differences were transformed to non-equidistant nite
differences according to Znidaric-Plazl and Plazl [18,27].
2.4. Prediction of diffusion coefcients
The correlation for protein diffusion coefcients D proposed by
He and Niemeyer [28] were used, which is based on the solutes
molecular weight M and radius of gyration RG and has shown
improvement in the estimation accuracy:

6:85  1015 T
q
l M1=s RG

where l is dynamic viscosity of the solvent in Pas and T is temperature in K. For BSA, M and RG are 66,000 Da and 28.7 , respectively
[28].
3. Experimental
3.1. Reagents
Bovine serum albumim (BSA) was purchased from Fluka (Bucks,
Switzerland), D-fructose from Merck (Hohenbrunn, Germany), 1butyl-3-methylimidazolium tetrauoroborate ([C4mim] [BF4])
from Io-li-tec (Heilbronn, Germany), while polyethylene glycol
(PEG) with molecular weight 4000 g/mol and KH2PO4 were from
SigmaAldrich (Steinheim, Germany).
3.2. Formation of ATPS and IL-ATPS
ATPS was prepared by mixing 16% (w/w) of PEG 4000, 11% (w/
w) K2HPO4 and 73% (w/w) of demineralized water in a 100 ml beaker. The mixture was mixed by means of a magnetic stirrer Rotamix 545 MMH (Tehtnica Zelezniki, Zelezniki, Slovenia) at
1000 rpm until all the components were dissolved and then transferred to a centrifuge tube. For ensuring the complete phase separation, centrifugation at 3000 rpm for 5 min was performed using
centrifuge LC 320 (Tehtnica Zelezniki, Zelezniki, Slovenia).

Data for the construction of binodal curve for the [C4mim]/Dfructose ATPS and for PEG 4000/KH2PO4 was taken from the literature [29,30]. Based on the binodal curve, the weight fractions of
phase forming components were used. IL-ATPS was prepared by
mixing different amounts of [C4mim] [BF4] 2664% (w/w) and
3674% (w/w) of 20% (w/w) D-fructose aqueous solution in test
tubes and stirred using vortex Vibromix 204 EV (Tehtnica Zelezniki, Zelezniki, Slovenia) at 2000 rpm for 3 min. The separation of
phases was achieved as in the case of ATPS. pH of the initial
20% (w/w) D-fructose-aqueous solution was adjusted with addition
of the aqueous solution of KOH or H3PO4. The pH values were measured with pH meter MA 5730 (Iskra, Kranj, Slovenia) with inlab
micro pH electrode (Mettler Toledo, Columbus, USA).
3.3. Determination of Kp for BSA in both two-phase systems
Partitioning coefcient Kp for BSA in ATPS composed of PEG/
pfosphate and in IL-ATPS composed of [C4mim] [BF4]/D-fructose
was determined by adding 1 g/l of BSA to the mixtures while forming both two-phase systems (Section 3.2). After centrifugation, the
two-phase systems were left for at least 12 h to achieve the equilibrium concentration of BSA. Samples from both phases were collected and analyzed for protein content as described below.
Partitioning coefcient was calculated as the ratio of the equilibrium concentrations of BSA in the top phase (phase 2) and in the
bottom phase (phase 1). At least three parallels were analyzed
and the mean values were calculated.
3.4. Determination of viscosities
An Ostwald viscosimeter which was thermostated at 25 C was
used for the determination of the viscosities of PEG-rich and phosphate-rich phases of ATPS formed as described previously [31].
3.5. Continuous extraction of BSA within a microchannel system
Extraction of BSA was carried out in glass microchannel systems
with y-shaped inow and outow channels (Micronit Microuidics
B.V., Enschede, The Netherlands) of two different geometries: the
rst with 220 lm, 50 lm and 664 mm and the second with
440 lm, 50 lm and 664 mm regarding width, height and length,
respectively. PEG/phosphate ATPS for BSA extraction was tested
in the rst system, while IL/D-fructose ATPS was tested in both
geometries. Tested ATPSs were prepared as specied in Section 3.3
and after phase separation 1 g/l of BSA was dissolved in phase 1
(phosphate-rich of ATPS or D-fructose-rich phase of IL-ATPS). As
shown in Fig. 1, phase 1 with BSA was fed from one microchannel
inlet and phase 2 (PEG-rich phase of ATPS or IL-rich phase of ILATPS) was fed from the other using syringes and high pressure syringe pumps (PHD 4400 Syringe Pump Series, Harvard Apparatus,
Holliston, USA). Both phases were supplied at constant ow rates
between 5 and 60 ll/min for phase 1 and between 8 and 90 ll/
min for phase 2, and collected at two outlets. All experiments were
performed at room temperature (25 C) in at least three parallels
and the mean values were calculated. The average dimensionless
concentration at the outlet of the microchannel X and extracted
fraction f were calculated as

c1;out
c1;in
f2  c2;out
f
f1  c1;in

5
6

where c1,in and c1,out are BSA concentrations in phase 1 at the

microchannel inlet and outlet, respectively, while c2,out is BSA

Please cite this article in press as: U. Novak et al., Ionic liquid-based aqueous two-phase extraction within a microchannel system, Separ. Purif. Technol.
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U. Novak et al. / Separation and Purication Technology xxx (2012) xxxxxx

concentration in phase 2 at the microchannel exit, whereas f1 and f2

are ow rates of phase 1 and phase 2, respectively.
3.6. Determination of BSA concentration
The concentration of BSA in the samples was determined spectrophotometrically at 595 nm by Bradford dye assay method [32]
(Cary 50, Varian Australia Pty. Ltd., Mulgrave, Australia). To avoid
interference from phase components, samples were analyzed
against blanks containing the same phase composition but without
proteins.
4. Results and discussion
4.1. The inuence of IL-ATPS composition and pH value on Kp
The mechanism governing the partition of proteins in ATPSs is
complex and still not fully understood. Generally, several factors
like type and concentration of the phase forming compounds, pH
and temperature of the ATPS are known to inuence the efciency
of the conventional PEG-based ATPSs [5].
Our studies on the effect of the IL concentration in IL-ATPS on
the partitioning coefcient for BSA at pH 7.3 revealed a trend of
Kp increasing up to the IL concentration of 46.7% (w/w), while at
higher [C4mim][BF4] concentrations, the partitioning of BSA into
the IL-rich phase decreased (Fig. 2a). A similar trend was observed
by Pei et al. [33] for BSA extraction efciency related to [C4mim]Br
concentration in a [C4mim]Br/phosphate ATPS, who concluded that
partition of proteins in IL-ATPS is driven by a combination of

hydrophobic interaction, electrostatic interactions and salting-out

effect. The study of Dreyer et al. [13] who used an IL-ATPS composed of an ionic liquid containing cation with oligoethyleneglycol
unit and an aqueous phosphate solution for the extraction of various proteins revealed that partitioning between phases mainly depends on molecular weight and the surface hydrophobicity of the
protein and also on electrostatic interaction between the charged
amino acid residues at a proteins surface and the positively
charged IL-cation [13]. However, the phenomenon of the formation
of IL aggregates and IL aggregate-BSA complexes in the IL-rich
phase was also suggested as a driving force for the selective separation of proteins and sugars in the [C4mim][N(CN)2]/K2HPO4 ATPS
[10]. By increasing IL concentrations, the number of IL-aggregate
complexes even increased [10], which obviously did not occur in
our case without an inorganic salt.
The inuence of the pH of starting D-fructose solution on Kp for
BSA in IL-ATPS was also studied at the same IL concentration of
47% (w/w). When using starting D-fructose solutions with pH below 6 and above 8.5, no separation of the phases could be achieved
after the addition of an adequate amount of the IL according to the
phase diagram [29]. Pei et al. also reported that [C4mim]Br/phosphate could not form two phases at pH below 6.0 [33]. Partition
of BSA into IL-rich phase was therefore studied between pH 6
and 8.5 and increased with the pH up to the value of 7.5, while further increase in pH of starting solution resulted in a decrease in Kp
(Fig. 2b). This is again a similar trend as for the cytochrome c
dependency of the pH of the extraction buffers in the [C4mim]Br/
phosphate ATPS [33]. Since the addition of IL decreased the pH of
the mixtures without a buffer, pH of the solution was probably closer to the isoelectric point of the protein, which for BSA is 4.8 [33],
where hydrophobic interactions are known to be the strongest
[34].
4.2. Continuous extraction in a microchannel

Fig. 2. Partitioning coefcient for BSA in IL-ATPS at 25 C as a function of (a)

[C4mim][BF4] concentration in IL-ATPS at pH 7.3 and (b) pH of a D-fructose-rich
phase at IL concentration of 46.7%.

4.2.1. Fluid ow pattern

The selected IL-based ATPS composed of 47% IL and with initial
D-fructose aqueous solutions pH of 7.5 was further used for protein extraction in glass microchannels with y-shaped entrance
and exit and further compared with the ATPS based on PEG/phosphate. As seen in Fig. 3ad, a liquidliquid interface parallel to the
sidewalls and in the middle of both microchannel geometries could
be formed at the proper ow ratio of both phases, enabling efcient phase separation at the y-shaped end of the microchannel.
It was demonstrated that relatively small differences in viscosity can have major effect on the relative distribution of two uid
streams within the microchannel system [35]. Based on the literature data for viscosities of both phases of IL-ATPS used in microchannels [36], as well as on our own viscosities determinations
for PEG/phosphate ATPS, which are summarized in Table 1, IL-rich
phase of [C4mim][BF4]/D-fructose IL-ATPS has almost 10 times lower dynamic viscosity when compared to PEG-rich phase in conventional PEG/phosphate ATPS, and also the relative viscosity of both
phases in the PEG/phosphate ATPS is 15, while in IL-ATPS it is 1.6.
Therefore, in order to achieve the position of the interface area
in the middle of the channel, presented in Fig. 3ad, the ratio of
ow rates for IL-rich phase and D-fructose-rich phase was 1.6:1,
while for the PEG-rich and phosphate-rich phase in ATPS, the ratio
of ow rates was 1:15. The effect of gravity in the microspace was
negligible compared to that of surface tension, and laminar two
phase ow could be stable in the side-by-side formation in spite
the density difference between both phases [37].
However, the experiments performed at ow rates below 5 ll/
min of D-fructose-rich or phosphate-rich phase revealed that the
system became unstable and it was not possible to separate both
phases at the microchannel exit. Therefore the experiments were

Please cite this article in press as: U. Novak et al., Ionic liquid-based aqueous two-phase extraction within a microchannel system, Separ. Purif. Technol.
(2012), doi:10.1016/j.seppur.2012.01.033

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Fig. 3. Photographs of a parallel ow of IL-ATPS at the entrance (a and c) and at the exit (b and d) of the 664 mm long microchannels of different geometries: (a and b) 220 and
50 lm and (c and d) 440 and 50 lm regarding width and depth, respectively.

Table 1
Measured and literature [36] data on dynamic viscosities and diffusivities for BSA in
all phases estimated at 25 C from Eq. (4).
Aqueous solution

l  103 (Pas)

D  1011 (m2/s)

D-Fructose rich phase

IL-rich phase
PEG-rich phase
Phosphate-rich phase

4.2 [36]

1.43

2.5 [36]
22.4
1.5

2.40
0.27
4.00

performed within the range of ow rates between 5 and 60 ll/min

for phase 1 and between 8 and 90 ll/min for phase 2.
It can be shown that at steady-state conditions a fully developed prole takes place very shortly after the beginning of the
microchannel [18]. Based on continuity and momentum equations
for a fully developed Poiseuille type ow [18,27], the fully developed 3D velocity proles for both ATPS were calculated and an
example is presented in Fig. 4. The rst simulation, shown in

Fig. 4a, was done for the description of the velocity prole in a
microchannel of 440 lm width with [C4mim][BF4]/D-fructose
based IL-ATPS, while the simulation presented in Fig. 4b represents
the channel of 220 width with PEG/phosphate ATPS. At these ow
rates, the average velocities at the interface of both phases were
0.0792 and 0.1034 m/s for ATPS and IL-ATPS, respectively.

4.2.2. Extraction within a microchannel system

The diffusion coefcients for BSA in all phases used in microchannel systems were calculated from Eq. (4) and are summarized
in Table 1. Partitioning coefcients Kp for BSA in IL-ATPS and in
PEG/phosphate ATPS were previously determined to be 14.6 and
0.734, respectively.
A fully developed velocity prole was used for the 3D simulation of BSA concentration prole within the whole microchannel,
considering the laminar nature of microuidic ows in co-ow
with the extracting uid so that molecules of BSA were able to diffuse into the phase 2. As it can be seen in Fig. 5, the experimental

Fig. 4. The developed velocity prole for two-phase parallel ow inside the microchannel (a) for [C4mim][BF4]/D-fructose based IL-ATPS in a microchannel, presented in
Fig. 3c and d, at f1 50 ll/min (average velocity in longitudinal direction vav = 0.0758 m/s) and f2 80 ll/min (vav = 0.1212 m/s) and (b) for PEG/phosphate ATPS in a
microchannel, presented in Fig. 3a and b, at f1 50 ll/min (vav = 0.1515 m/s) and f2 4 ll/min (vav = 0.0121 m/s).

Please cite this article in press as: U. Novak et al., Ionic liquid-based aqueous two-phase extraction within a microchannel system, Separ. Purif. Technol.
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Fig. 5. The model simulations and experimental values of average dimensionless concentration of BSA in the phase 1 at the exit of the microchannel, presented in Fig. 3a and
b, and extracted fraction of BSA in continuous extraction process at different retention times of the phase 2(sphase2) in the same microchannel system using IL-ATPS and PEG/
phosphate ATPS.

yields of extraction in the micro-ow system were very close to the

values of mathematical model calculations. The highest achieved
extraction efciency of BSA from D-fructose rich phase to IL-rich
phase in a microchannel was 53% at both used aspect ratios. Due
to the difculties with the ow stability at lower ow rates longest
possible retention times in the used microchannel systems for the
D-fructose-rich phase was 110 s and for IL-rich phase 175 s.
According to simulations, the use of a longer channel would be
benecial for better extraction yields.
The results were compared to PEG/phosphate ATPS, where the
highest extraction efciency for BSA reached in the tested microchannel, presented in Fig. 3a and b, was 6% (Fig. 5). This conrmed
the benet of using IL-ATPSs for the extraction of biomolecules as
compared to conventional PEG/salt ATPS, primarily due to the
unfavorable relative viscosity of phases in the latter. Estimated
productivities of this microchannel system at the highest extraction fractions obtained at our experimental conditions were 17.4
and 1.48 kg/h m3 for IL-ATPS and PEG-based ATPS, respectively.
For increasing the capacity of the system, a numbering-up approach by using parallel microextractors is proposed.
5. Conclusions
In the present work, continuous extraction of protein in different aqueous two-phase systems was carried out in microchannel
systems enabling the separation of both phases at the exit. ILbased ATPS exhibited several advantages when compared with
the conventional PEG/phosphate ATPS such as lower viscosity, little emulsion formation and quick phase separation. Especially lower viscosities of both phases were benecial for the process
feasibility in a microchannel system. The highest extracted fraction
obtained was 53% at the longest possible retention time of 175 s
which still enabled the continuous separation of both phases at
the microchannel exit. Therefore for longer available retention
time, the use of longer and/or consecutively bounded microchannel system would be benecial. For the increasing of the system
capacity, a numbering-up approach by using parallel microextractors is proposed.
However, the successful future application of IL-ATPSs also depends on the ecotoxicity of the ILs. Although the activity of biomacromolecules is maintained in the IL-rich phases of IL-ATPSs,
the wide application of such separation techniques would unavoidably result in the loss of the ILs into water ecosystems, leading to

environmental pollution, so non-toxic or biocompatible ILs (e.g.

choline [38] and amino-acid based ILs [3942]) will gain further
attention to develop environmentally benign IL-ATPSs. Since acidic
conditions or high temperatures promote the anion hydrolysis of
hexauorophosphate-based ILs in aqueous solutions [43], further
screening of more environmentally benign IL-ATPSs is envisaged.
The use of microuidic devices such as suggested in this work
could serve as an efcient tool for the screening of these solvents
and for process parameter evaluation in order to obtain highly efcient and environment-friendly continuous extraction systems.

Acknowledgement
The nancial support of the Ministry of Higher Education, Science and Technology of the Republic of Slovenia through Grant
P2-0191, PhD Grant 1000-10-310199 (U. Novak) and PhD Grant
1000-07-310011 (A. Pohar) is gratefully acknowledged.

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Please cite this article in press as: U. Novak et al., Ionic liquid-based aqueous two-phase extraction within a microchannel system, Separ. Purif. Technol.
(2012), doi:10.1016/j.seppur.2012.01.033