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Droplet-based microextraction in the aqueous two-phase system
Journal of Chromatography A, 1217 (2010) 3723–3728
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Droplet-based microextraction in the aqueous two-phase system Young Hoon Choi, Young Soo Song, Do Hyun Kim ∗ Department of Chemical and Biomolecular Engineering and Center for Ultramicrochemical Process Systems, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea
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Article history: Received 22 October 2009 Received in revised form 3 April 2010 Accepted 9 April 2010 Available online 4 May 2010 Keywords: Microextraction Aqueous two-phase system (ATPS) Electrohydrodynamics Droplet
a b s t r a c t This report is about microfluidic extraction systems based on droplets of aqueous two-phase system. Mass transfer between continuous phase and dispersed droplet is demonstrated by microextraction of ruthenium red in a microfluidic device. Droplets are generated with electrohydrodynamic method in the same device. By comparing brightness in the digital image of a solution with known concentrations of ruthenium red and those of a droplet in the microextraction, ruthenium red concentration was measured along the microextraction channel, resulting in good agreement with a simple diffusion model. The maximum partition coefficient was 9.58 in the experiment with the 70-mm-long-channel microextractor. The method is usable for terminating microextraction by electrohydrodynamic manipulation of droplet movement direction. Droplets of different ruthenium red concentration, 0.12 and 0.24% (w/w) in this experiment, can be moved to desired place of microfluidic system for further reaction through respectively branched outlets. In this study droplet-based microextraction is demonstrated and the mass transport is numerically analyzed by solving the diffusion–dissolution model. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Recent studies on droplet-based microfluidics have drawn great attentions with the development of new methods and applications in various fields of lab-on-a-chip technology. Droplets provide unique opportunities to handle fluids in a discrete manner, offering novel platform of miniaturized system for chemical and biological processes [1]. Compartmentalization of pre-defined fluid in an immiscible phase system allows enhanced reaction with reduced dispersion of reactants, taking the advantages of mixing by recirculation and fast molecular transport across the interface between disperse and continuous phase due to the high interface-areato-volume ratio. In addition, possible control of droplet breakup, coalescence and movement by various physical forces leads to individual and independent use of droplets as tiny and convenient reaction vessels and fluid carriers in a microfluidic system. These features of droplet-based microfluidics have been beneficial in areas such as biology, drug discovery and chemical synthesis [2–5]. Two common techniques for generation of droplet in microfluidic system are dispersing fluid in a continuous phase with the configuration of T-junction and flow-focusing. Depending mainly on the geometry of the microchannels, flow rate combination and relative viscosity of the fluids, the disperse phase is elongated and eventually broken into droplets of small volume [6–8]. Electrohy-
∗ Corresponding author. Tel.: +82 42 350 3929; fax: +82 42 350 3910. E-mail address: [email protected] (D.H. Kim). 0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2010.04.015
drodynamic (EHD) method is also employed in generating droplets of fluid with the application of electric field across the electrodes installed in the microfluidic system. The advantage of EHD dropletgeneration methods that require no external pumps lies on the fact that the same EHD principle can be used to manipulate and control the generated droplets, and to transport them to desired part of the microfluidic system at desired moment for further processing during the analysis and reaction. The feasibility of EHD droplet generation in microfluidic systems is supported by many recent reports [9–13]. Most of the reports on microfluidic droplet generation and applications are based on organic/aqueous two-phase system. One of the drawbacks in using organic solvents is the incompatibility with the applications dealing with biological molecules including proteins, amino acids and others. Aqueous two-phase system (ATPS) can be an appealing system for microfluidic droplet application, since both phases are aqueous [14,15]. ATPS consists of two immiscible phases formed by dissolving two incompatible polymers, such as poly(ethylene glycol) and dextran, or one polymer and an appropriate inorganic salt, or a cationic surfactant and a salt. ATPS is highly advantageous because the high water content (usually 70–90% (w/w)) provides biocompatibility and selectivity for hydrophilic molecules and interfacial tension is relatively low compared to that of organic/aqueous two-phase system. It can be a versatile partitioning system for the separation of many kinds of dyes, metal ions, silica particles, proteins and cells [15,16]. Recently, ATPS has started to be employed for the application in microfluidic systems. Continuous partitioning of cells in the poly(ethylene glycol)/dextran aqueous two-phase flow system has
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been reported by Yamada et al. [17] and Nam et al. [18]. Münchow et al. [19–21] reported electrophoretic partitioning of proteins by the diffusive transport of proteins across a fluid phase boundary in the ATPS. Meagher et al. [22] developed a process in a microscale device for isolating specific proteins from Escherichia coli cell lysate based on the single-stage poly(ethylene glycol)–salt ATPS. Our group has extended the application of ATPS from stratified flow system to EHD generation and manipulation of ATPS droplets [12,13]. Microextraction is one of the representative unit operations using two immiscible liquid phases in microfluidic systems. In addition to the work of Kitamori’s group [23] on continuous solvent extraction, droplet-based extraction in microfluidic system has also been reported. Xu et al. [24] used flow of droplets to enhance the mass transfer in the microfluidic device and demonstrated extraction of succinic acid from n-butanol to aqueous droplets containing sodium hydroxide. Mary et al. [25] reported the extraction of fluorescein or rhodamine B between droplets of aqueous phase and the external phase of octan-1-ol, in a PDMS microfluidic device. Castel et al. [26] reported continuous molecular enrichment in a microfluidic system aided by the vortex within segmented droplets. In our experiment, we demonstrate microextraction based on droplets of ATPS which is formed by dissolving tetrabutylammonium bromide (TBAB) and ammonium sulfate as prepared by Akama et al. [27]. Though much lower than that of organic/aqueous two-phase system, interfacial tension in this ATPS is relatively high (about 4–5 mN/m) compared to that of common ATPS formed with poly(ethylene glycol) and dextran. Thus the interface readily responds to the external electric field in microchannels. Taking the advantage of the EHD generation and transport of droplets, we demonstrate the microextraction of ruthenium red from continuous phase to dispersed droplets and its termination by droplet movement control.
Fig. 1. (a) The schematic diagram of the microfluidic system for the analysis of kinetics of microextraction of ruthenium red. (b) The schematic diagram of the microfluidic system for microextraction control. Droplets can be switched to one of the two branched channels by applied electric pulse for the termination of microextraction.
2. Experimental 2.1. Microextraction device fabrication Microfluidic devices made of poly(dimethylsiloxane) (PDMS) (Sylgard 184, Dow Corning, Midland, MI, USA) and glass were fabricated by soft lithography technique [28] which includes spincoating of photoresist on silicon wafer and patterning by exposure to UV light using transparency film photomask. A negative photoresist (SU-8 2100, MicroChem, Newton, MA, USA) was used to make the structure with a thickness of 100 m. For molding of the replica structure of developed photoresist, PDMS prepolymer was mixed with crosslinker (mass ratio 10:1) and degassed. Then the mixture was poured onto the photoresist structure and cured for 2 h at 70 ◦ C. The cured PDMS replica was peeled off the photoresist structure and bonded with a flat slide glass which was pre-treated with corona discharge after carefully making holes of same size for the liquid inlets and outlets. Thus, the resulting microfluidic device has PDMS for the walls and the top, and glass for the bottom. Stainless steel tubes, which serve as both electrodes and inlet or outlet ports, were inserted through the holes and connected with polyethylene tubes (Intramedic, Becton Dickinson, Sparks, MD, USA). 2.2. ATPS preparation ATPS used in this experiment was prepared by dissolving TBAB (MW 322.38, 99%, Sigma–Aldrich, St. Louis, MO, USA) and ammonium sulfate (MW 132.14, >99.0%, Sigma–Aldrich) in deionized water as suggested by Akama et al. [27]. The solution of TBAB and ammonium sulfate was vigorously mixed for 1 h and settled at least for 3 h until stable and clear interface was observed. The concentrations of TBAB and ammonium sulfate were 15% and 30% (w/w),
respectively, prepared by dissolving 5.45 g and 10.9 g of each in 20 g of deionized water. Each phase was taken individually and fed into the microextraction device with syringe pumps (KD Scientific, Holliston, MA, USA) that are operated individually. The TBAB-rich phase, which has lower density and higher viscosity, serves as the continuous phase while the ammonium sulfate-rich phase is a disperse phase to form droplets. Deformation of interface, movement of droplet and color change inside droplets were observed by microscope (SZX-ILLB2-200, Olympus, Tokyo, Japan) and recorded by digital camera (C-5060, Olympus). 2.3. Droplet-based microextraction process As shown in the schematic diagrams of droplet-based microextraction process in Fig. 1a and b, the microextraction device has two parts: Droplet-generation part and microextraction part. TBAB-rich phase and ammonium sulfate-rich phase are introduced through the inlets in droplet-generation part and their flow rates are maintained to form stable laminar flow. Potential difference is applied between the electrodes located at the end of T-branches by d.c. power supply (PNCYS-1501, PNCYS, Uiwang-si, South Korea) capable of potential difference up to 150 V. This potential difference of 150 V was applied as pulsed signal of 200 ms generated with LabView software (National Instruments, Austin, TX, USA). Generated droplets move to the second part of the microextraction device which has another inlet for the injection of the TBABrich phase containing ruthenium red (MW 786.36, >85%, Fluka, Buchs, Germany) that has the structural formula of [(NH3 )5 Ru-ORu(NH3 )4 -O-Ru(NH3 )5 ]Cl6 . The produced droplets are swept into the new stream by the shear caused by velocity difference and the microextraction begins in the new stream. The microextrac-
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Fig. 2. Correlation between the brightness from the digital images of ammonium sulfate-rich droplets and ruthenium red concentrations. The digital images in the box on the right show the difference in brightness of the ammonium sulfate-rich phase droplets whose ruthenium red concentrations are 0%, 0.1%, 0.4%, 0.7%, 1.0% and 1.3% (w/w). The expression for the straight line obtained by linear regression is: (concentration) = − 0.013 × (brightness) + 2.37. R2 = 0.9871.
tion device for microextraction control has two branched channels at the position of 10 and 20 mm from the junction of the ruthenium red injection where microextraction begins as shown in Fig. 1b. Microextraction continues until the pulse signal is applied at one of the outlet branches and the droplets are moved into switched to that branch. The concentration of ruthenium red inside the ammonium sulfate-rich droplet at every 10 mm was measured by the analysis of brightness data from digital images. Using the normalized brightness data of the digital images collected by Scion Image software (Scion, Frederick, MD, USA), the concentration of ruthenium red extracted into the ammonium sulfate-rich droplets was analyzed by Photoshop 7 (Adobe, San Jose, CA, USA), after calibrating the data earned from microscopic images of ammonium sulfaterich droplets with known concentrations of ruthenium red. As shown in Fig. 2, brightness data were normalized based on the six ammonium sulfate-rich phase droplets of which concentrations of ruthenium red are 0, 0.1%, 0.4%, 0.7%, 1.0% and 1.3% (w/w), respectively, and the correlation between concentration of ruthenium red and brightness was constructed. Although this is not a direct concentration measurement by spectroscopy, it allows the acquisition of concentration data in situ in the microfluidic device during microextraction. It can avoid any further mass transfer between the droplet and the continuous phase, which is possible in the case of direct sampling of droplets. We collected the brightness data, not from the whole droplet, but within the circle whose radius is 0.7R (R denotes the radius of the whole droplet) not to include the edge rim of the droplet because brightness may not be correct due to the presence of interface. To determine the concentration, the collected brightness information was compared with the baseline brightness value that is the integrated brightness data in the PDMS wall area of the digital image. The data were averaged with at least 10 droplet images that were taken at the same time of the microextraction process. 3. Results and discussion 3.1. Droplet generation A droplet is electrohydrodynamically generated at the ‘dropletgeneration’ part of each device in Fig. 1a and b. TBAB-rich phase and ammonium sulfate-rich phase are introduced individually through the inlets and contacted with each other at the Y-junction, forming laminar flow along the main microchannel. At the T-junction, entire ammonium sulfate-rich phase with smaller viscosity and a
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portion of TBAB-rich phase flow together through one branch of the channel, while the other branched channel at the T-junction is filled with TBAB-rich phase only. When a pulse of d.c. electric potential is applied between the electrodes located at the end of the branched channels, ammonium sulfate-rich phase is attracted toward the positive electrode and the interface is deformed drastically, to dispense a small volume of ammonium sulfate-rich phase as a droplet dispersed in TBAB-rich phase. Details of the droplet generation and its mechanism can be found in our previous reports [12,13]. For droplet generation, several different EHD forces are thought to be acting in combination. While inertia of streams may suppress the generation of droplets, the electrostatic attraction of ammonium sulfate-rich phase toward the positive electrode is the primary force that deforms the interface and generates droplets. The interfacial tension also helps the generation of ammonium sulfate-rich droplets, minimizing the interfacial energy. Once the ammonium sulfate-rich phase stream is elongated by the electric field, the shear force of the TBAB-rich phase stream chops the ammonium sulfaterich phase stream into droplets. The perpendicular channel wall at the T-junction is also essential as a ‘chopping board’ for splitting the extended ammonium sulfate-rich phase stream into droplets. In our experiments, higher voltage is required to generate even a single droplet, when the angle between the main microchannel and branched channel connected to the positive electrode is larger than a right angle. Although the TBAB-rich phase is more hydrophobic than ammonium sulfate-rich phase so that the former phase had constant contact with the PDMS surface which is hydrophobic as well, both phases are basically hydrophilic due to the high water content. In addition, since the TBAB-rich phase is the continuous phase in which ammonium sulfate-rich phase is dispersed, the more hydrophilic nature of the ammonium sulfate-rich phase did not have great effects on the droplet movement in our experiment. The mean radius of the droplets in the experiment was 58.4 m and the standard deviation was 3.8 m. 3.2. Microextraction kinetics To understand microextraction kinetics of ATPS, we employed a microchannel of the length of 70 mm in the extraction part shown in Fig. 1a. This length is sufficient to allow microextraction to continue until color change in droplets is no longer noticed by microscopic observation. The feed flow rates of ammonium sulfate-rich phase and TBAB-rich phase into droplet-generation part are kept at 5.04 l/min and 0.14 l/min, respectively. In the microextraction part, ruthenium red-containing TBAB-rich phase is introduced to the main microextraction channel, as shown in Fig. 1a and b schematically, at the flow rate of 0.24 l/min. The flow rate combination is selected carefully to have appropriate pressure drop and width of each stream, especially at the T-junction in droplet-generation part and at the junction where ruthenium red stream is introduced in microextraction part. Six kinds of ruthenium red-containing TBAB-rich phases were prepared by dissolving ruthenium red in the TBAB-rich phase with the concentration of 0.05%, 0.07%, 0.09%, 0.11%, 0.13% and 0.15% (w/w). Ruthenium red was dispersed in the TBAB-rich phase as solid particles, but was highly soluble in ammonium sulfate-rich phase as identified by its color. We assume the whole extraction process to be composed of two different steps that are clearly observed with optical microscope. One is the process where ruthenium red in the continuous phase is transported to the disperse phase and dissolved on the surface of the ammonium sulfate-rich phase droplet until it is saturated. The general equation for the process involving dissolution of dispersed solid particles can be written as [29,30] ∂Cs = kdiss (Csat − Cs ) ∂t
(1)
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where Cs denotes the concentration at the surface of the droplet, Csat , the concentration when saturated, and kdiss , the dissolution rate constant. Integrating above equation gives Cs as a function of time as follows: Cs = Csat (1 − exp(−kdiss t))
(2)
The second step is the transfer of ruthenium red from the surface of droplet to the center of the droplet. For this step, onedimensional model of simple diffusion with spherical coordinate system is ∂Cin 1 ∂ =D 2 ∂t r ∂r
r
2 ∂Cin
(3)
Fig. 3. Comparison of the experimental (symbols) and simulated data (lines) of the droplet-based microextraction kinetics.
where Cin and D denote the concentration of solute inside the droplet and the effective diffusion coefficient, respectively. This model assumes no internal motion of the fluid in the droplet. The concentration in the continuous phase is assumed to be constant because TBAB-rich phase with fixed concentration of ruthenium red is continuously fed to the microextraction device. The boundary conditions at r = 0 and r = R for this equation are specified as
are shown in Fig. 3. Here, Cout is the concentration of ruthenium red in the continuous TBAB-rich phase. Time-dependent simulation results are in good agreement with the experimental results except the lowest concentration results (Cout = 0.05% (w/w)) in their initial time stage where simulation overestimates the concentrations. One explanation for this error may be that in the early stages of microextraction, the ruthenium red is mainly in the region near the surface of the droplet which is excluded in the brightness measurement especially when the concentration of ruthenium red is very low. As the extraction process continues, the more ruthenium red is diffused to the region belonging to the brightness measurement, and simulation shows better agreement with experimental result. Fig. 4 shows the ratio of the concentration of ruthenium red in the droplet after 45 s of microextraction to that of continuous TBABrich phase fed initially. In the first three cases of low ruthenium red concentrations (0.05%, 0.07% and 0.09% (w/w)) in TBAB-rich phase, the ratio is almost constant. However, the ratio decreases, as the concentration of ruthenium red of the TBAB-rich phase increases for high range of concentration of ruthenium red. This suggests that there exists the solubility limit of ruthenium red in the ammonium sulfate-rich droplet above which the concentration cannot increase independent of the concentration in the TBAB-rich phase. The solubility limit of ruthenium red in the ammonium sulfate-rich phase droplet, corresponding to Cout of 0.13% and 0.15% (w/w), is about 1.15% (w/w) in this experiment.
∂r
,
∂Cin = 0 at r = 0, ∂r
(4)
Cin = Cs = Csat (1 − exp(−kdiss t))
at r = R.
(5)
The initial condition is Cin = 0 at t = 0
for all r.
(6)
The first boundary condition (Eq. (4)) assumes symmetry at the center of the droplet. In the second boundary condition (Eq. (5)), Csat is the concentration on the surface of the droplet at saturation, which corresponds to the product of concentration of ruthenium red in TBAB-rich phase and distribution coefficient between two phases. Although concentration in the TBAB-rich phase is known and assumed to be constant, the distribution coefficient is not available. After sufficiently long extraction time, the droplet will be saturated with solute and the concentration will correspond to Csat . In this work, we assume the extraction time is long enough and the final concentration at the end of extraction is considered to be Csat . Solving the equation numerically with the boundary conditions gives radial distribution of concentration in the droplet at each time of concentration measurement. The average concentration in a spherical region with a radius of 0.7R inside a droplet is obtained by
2 0.7R
Cavg =
0
0
o
Cr 2 sin drdd
2 0.7R 0
0
o
r 2 sin drdd
.
3.3. Microextraction control For the facility in the operation of the process, the extraction time is shown to be controlled by guiding the droplet movement with EHD force. The time-dependent profile of microextraction
(7)
In this equation, interval of integration for r is 0 ≤ r ≤ 0.7R to be consistent with the experimental measurement as explained in the experimental section. To solve the equation numerically, we need information about Csat , kdiss and D. The measured concentration of ruthenium red at 70 mm position which is the end of microchannel was chosen as Csat in each experiment. The kdiss value was determined as 0.09 (s−1 ) by fitting experimental results from the entire microextraction process and applied to solve the equation with five different Csat . (We have used the same Csat for experiments of 0.13% and 0.15% (w/w) assuming that the Csat has reached its solubility limit. The increase of the ruthenium red concentration in TBAB-rich phase from 0.13% to 0.15% did not cause further increase in the concentration in the droplet. So, we assumed that the concentration of 1.15% might be the final concentration which is the solubility limit.) Diffusion coefficient of ruthenium red in ammonium sulfate-rich phase, D, was measured by using ‘T-sensor’ developed by Kamholz et al. [31]. Solutions of simple diffusion model with experimental results
Fig. 4. Ratio of the ruthenium red concentration in the droplet after 45 s of microextraction to that of continuous TBAB-rich phase initially introduced to the microextraction device. The lines indicate that the concentration could reach the solubility limit, not increasing continuously.
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Fig. 5. Microextraction control and manipulation of droplet within the microextraction system shown in Fig. 1b. (a) Beginning of microextraction: The ammonium sulfaterich phase droplet enters the ruthenium red-containing TBAB-rich phase at the bottleneck of the microchannel and microextraction starts. (b) The first branched channel at the position of 10 mm from the starting point of microextraction. In this case, the electric pulse is not applied so that the droplet keeps moving through the microextraction channel. (c) The second branched channel at the position of 20 mm from the starting point of microextraction. Applied electric pulse changes the direction of movement of the droplet toward the branched channel so that the microextraction is terminated and the droplet is guided to other part of the microfluidic system.
process obtained from the experimental and simulation results in Section 3.2, together with carefully designed microextraction device and droplet-motion guiding technique, allow us to determine the time needed for a droplet to reach a certain concentration which can be utilized for the specific research or analysis. The microextraction device for this experiment has two branched channels at the position of 10 and 20 mm from the junction of the ruthenium red injection where microextraction begins as shown in Fig. 1b. The branched channels were arranged to have an angle of 45◦ so that droplets can readily move toward the branched channels. Ammonium sulfate-rich phase that forms laminar flow with TBAB-rich phase in the droplet-generation part of the device is fed at the flow rate 5.4 l/min. The droplet produced in the dropletgeneration part of the device moves to microextraction part, and then extraction starts at the junction where TBAB-rich phase with ruthenium red concentration of 0.29% (w/w) is injected. Ruthenium red favors the ammonium sulfate-rich phase in bulk equilibrium state when the two phases are in contact. Only when the droplet crosses the streamlines and is fully surrounded by the ruthenium red-containing TBAB-rich phase, isotropy of microextraction is guaranteed. To force droplet to enter the ruthenium red stream, we fabricated the microchannel to have a narrow cross-section as shown in Fig. 5a. Because the flow rate of the main TBAB-rich phase, which initially carries the ammo-
nium sulfate-rich droplet, is kept at 0.24 l/min, smaller than that of ruthenium red solution (0.51 l/min), the droplet moves toward the ruthenium red solution due to the pressure difference. Microextraction continues until the d.c. electric pulse with the duration of 200 ms is applied at the end of the branched channel. At that moment the droplet departs ruthenium red-containing TBAB-rich phase by approaching the electrode located at each end of the branched microchannel and microextraction is terminated. As shown in Fig. 5b and c, the thresholds located at 10 and 20 mm positions serve as leaping boards for the droplet. When d.c. electric potential is not applied, the droplet moves freely following its streamline over the threshold as shown in Fig. 5b. When electric potential is applied on the electrode at the end of the branched channel, the droplet changes its direction at the junction so that it leaves the flow stream of ruthenium red-containing TBAB-rich phase and jumps into the branched channel with increased speed due to the electrophoretic effect as shown in Fig. 5c. The ruthenium red concentrations of droplets at the first and second branched microchannel when microextraction is terminated are 0.12% and 0.24% (w/w) and corresponding concentrations which have been predicted by simulation are 0.11% and 0.28% (w/w), which shows that the droplet-based microextraction can be terminated depending on the needs in a process, even before reaching the equilibrium state.
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4. Conclusions
References
Droplet-based microextraction with liquid–liquid ATPS has been investigated in this work. The microfluidic devices for EHD generation of aqueous two-phase droplet and microextraction were integrated into a single system which includes simple d.c. electric potential application and programmed control of droplet movement. Solving the equations of simple mass balance and one-dimensional diffusion in spherical coordinate for each step of the microextraction process with appropriate boundary conditions gave solutions that are in good agreement with the experimental data. For the facility in operation, the technique to stop microextraction process was demonstrated by changing the droplet movement direction based on the time-dependent microextraction profile that was investigated by the experiments. We have also tried to predict the concentration by the numerical solution of the simple model of the two-step molecular transport involving diffusion and dissolution. These results suggest that a single microfluidic system can be designed and fabricated to produce droplets with desired solute concentrations. Droplets of known concentrations of certain solute can be further used in the same microfluidic system for analysis, reaction and other purposes if the solute is preferentially distributed in the dispersed phase of the ATPS. From our experiences, the solutes can be inorganic particles, metal ions, and chelating agents. A wider range of application of ATPS droplet-based microfluidic system reported in this paper is expected to take the full advantage of the benefits that the system can provide.
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Acknowledgement This work was supported by Center for Ultramicrochemical Process Systems sponsored by Ministry of Education, Science and Technology.
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Droplet-based microextraction in the aqueous two-phase system Young Hoon Choi, Young Soo Song, Do Hyun Kim ∗ Department of Chemical and Biomolecular Engineering and Center for Ultramicrochemical Process Systems, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea
a r t i c l e
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Article history: Received 22 October 2009 Received in revised form 3 April 2010 Accepted 9 April 2010 Available online 4 May 2010 Keywords: Microextraction Aqueous two-phase system (ATPS) Electrohydrodynamics Droplet
a b s t r a c t This report is about microfluidic extraction systems based on droplets of aqueous two-phase system. Mass transfer between continuous phase and dispersed droplet is demonstrated by microextraction of ruthenium red in a microfluidic device. Droplets are generated with electrohydrodynamic method in the same device. By comparing brightness in the digital image of a solution with known concentrations of ruthenium red and those of a droplet in the microextraction, ruthenium red concentration was measured along the microextraction channel, resulting in good agreement with a simple diffusion model. The maximum partition coefficient was 9.58 in the experiment with the 70-mm-long-channel microextractor. The method is usable for terminating microextraction by electrohydrodynamic manipulation of droplet movement direction. Droplets of different ruthenium red concentration, 0.12 and 0.24% (w/w) in this experiment, can be moved to desired place of microfluidic system for further reaction through respectively branched outlets. In this study droplet-based microextraction is demonstrated and the mass transport is numerically analyzed by solving the diffusion–dissolution model. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Recent studies on droplet-based microfluidics have drawn great attentions with the development of new methods and applications in various fields of lab-on-a-chip technology. Droplets provide unique opportunities to handle fluids in a discrete manner, offering novel platform of miniaturized system for chemical and biological processes [1]. Compartmentalization of pre-defined fluid in an immiscible phase system allows enhanced reaction with reduced dispersion of reactants, taking the advantages of mixing by recirculation and fast molecular transport across the interface between disperse and continuous phase due to the high interface-areato-volume ratio. In addition, possible control of droplet breakup, coalescence and movement by various physical forces leads to individual and independent use of droplets as tiny and convenient reaction vessels and fluid carriers in a microfluidic system. These features of droplet-based microfluidics have been beneficial in areas such as biology, drug discovery and chemical synthesis [2–5]. Two common techniques for generation of droplet in microfluidic system are dispersing fluid in a continuous phase with the configuration of T-junction and flow-focusing. Depending mainly on the geometry of the microchannels, flow rate combination and relative viscosity of the fluids, the disperse phase is elongated and eventually broken into droplets of small volume [6–8]. Electrohy-
∗ Corresponding author. Tel.: +82 42 350 3929; fax: +82 42 350 3910. E-mail address: [email protected] (D.H. Kim). 0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2010.04.015
drodynamic (EHD) method is also employed in generating droplets of fluid with the application of electric field across the electrodes installed in the microfluidic system. The advantage of EHD dropletgeneration methods that require no external pumps lies on the fact that the same EHD principle can be used to manipulate and control the generated droplets, and to transport them to desired part of the microfluidic system at desired moment for further processing during the analysis and reaction. The feasibility of EHD droplet generation in microfluidic systems is supported by many recent reports [9–13]. Most of the reports on microfluidic droplet generation and applications are based on organic/aqueous two-phase system. One of the drawbacks in using organic solvents is the incompatibility with the applications dealing with biological molecules including proteins, amino acids and others. Aqueous two-phase system (ATPS) can be an appealing system for microfluidic droplet application, since both phases are aqueous [14,15]. ATPS consists of two immiscible phases formed by dissolving two incompatible polymers, such as poly(ethylene glycol) and dextran, or one polymer and an appropriate inorganic salt, or a cationic surfactant and a salt. ATPS is highly advantageous because the high water content (usually 70–90% (w/w)) provides biocompatibility and selectivity for hydrophilic molecules and interfacial tension is relatively low compared to that of organic/aqueous two-phase system. It can be a versatile partitioning system for the separation of many kinds of dyes, metal ions, silica particles, proteins and cells [15,16]. Recently, ATPS has started to be employed for the application in microfluidic systems. Continuous partitioning of cells in the poly(ethylene glycol)/dextran aqueous two-phase flow system has
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been reported by Yamada et al. [17] and Nam et al. [18]. Münchow et al. [19–21] reported electrophoretic partitioning of proteins by the diffusive transport of proteins across a fluid phase boundary in the ATPS. Meagher et al. [22] developed a process in a microscale device for isolating specific proteins from Escherichia coli cell lysate based on the single-stage poly(ethylene glycol)–salt ATPS. Our group has extended the application of ATPS from stratified flow system to EHD generation and manipulation of ATPS droplets [12,13]. Microextraction is one of the representative unit operations using two immiscible liquid phases in microfluidic systems. In addition to the work of Kitamori’s group [23] on continuous solvent extraction, droplet-based extraction in microfluidic system has also been reported. Xu et al. [24] used flow of droplets to enhance the mass transfer in the microfluidic device and demonstrated extraction of succinic acid from n-butanol to aqueous droplets containing sodium hydroxide. Mary et al. [25] reported the extraction of fluorescein or rhodamine B between droplets of aqueous phase and the external phase of octan-1-ol, in a PDMS microfluidic device. Castel et al. [26] reported continuous molecular enrichment in a microfluidic system aided by the vortex within segmented droplets. In our experiment, we demonstrate microextraction based on droplets of ATPS which is formed by dissolving tetrabutylammonium bromide (TBAB) and ammonium sulfate as prepared by Akama et al. [27]. Though much lower than that of organic/aqueous two-phase system, interfacial tension in this ATPS is relatively high (about 4–5 mN/m) compared to that of common ATPS formed with poly(ethylene glycol) and dextran. Thus the interface readily responds to the external electric field in microchannels. Taking the advantage of the EHD generation and transport of droplets, we demonstrate the microextraction of ruthenium red from continuous phase to dispersed droplets and its termination by droplet movement control.
Fig. 1. (a) The schematic diagram of the microfluidic system for the analysis of kinetics of microextraction of ruthenium red. (b) The schematic diagram of the microfluidic system for microextraction control. Droplets can be switched to one of the two branched channels by applied electric pulse for the termination of microextraction.
2. Experimental 2.1. Microextraction device fabrication Microfluidic devices made of poly(dimethylsiloxane) (PDMS) (Sylgard 184, Dow Corning, Midland, MI, USA) and glass were fabricated by soft lithography technique [28] which includes spincoating of photoresist on silicon wafer and patterning by exposure to UV light using transparency film photomask. A negative photoresist (SU-8 2100, MicroChem, Newton, MA, USA) was used to make the structure with a thickness of 100 m. For molding of the replica structure of developed photoresist, PDMS prepolymer was mixed with crosslinker (mass ratio 10:1) and degassed. Then the mixture was poured onto the photoresist structure and cured for 2 h at 70 ◦ C. The cured PDMS replica was peeled off the photoresist structure and bonded with a flat slide glass which was pre-treated with corona discharge after carefully making holes of same size for the liquid inlets and outlets. Thus, the resulting microfluidic device has PDMS for the walls and the top, and glass for the bottom. Stainless steel tubes, which serve as both electrodes and inlet or outlet ports, were inserted through the holes and connected with polyethylene tubes (Intramedic, Becton Dickinson, Sparks, MD, USA). 2.2. ATPS preparation ATPS used in this experiment was prepared by dissolving TBAB (MW 322.38, 99%, Sigma–Aldrich, St. Louis, MO, USA) and ammonium sulfate (MW 132.14, >99.0%, Sigma–Aldrich) in deionized water as suggested by Akama et al. [27]. The solution of TBAB and ammonium sulfate was vigorously mixed for 1 h and settled at least for 3 h until stable and clear interface was observed. The concentrations of TBAB and ammonium sulfate were 15% and 30% (w/w),
respectively, prepared by dissolving 5.45 g and 10.9 g of each in 20 g of deionized water. Each phase was taken individually and fed into the microextraction device with syringe pumps (KD Scientific, Holliston, MA, USA) that are operated individually. The TBAB-rich phase, which has lower density and higher viscosity, serves as the continuous phase while the ammonium sulfate-rich phase is a disperse phase to form droplets. Deformation of interface, movement of droplet and color change inside droplets were observed by microscope (SZX-ILLB2-200, Olympus, Tokyo, Japan) and recorded by digital camera (C-5060, Olympus). 2.3. Droplet-based microextraction process As shown in the schematic diagrams of droplet-based microextraction process in Fig. 1a and b, the microextraction device has two parts: Droplet-generation part and microextraction part. TBAB-rich phase and ammonium sulfate-rich phase are introduced through the inlets in droplet-generation part and their flow rates are maintained to form stable laminar flow. Potential difference is applied between the electrodes located at the end of T-branches by d.c. power supply (PNCYS-1501, PNCYS, Uiwang-si, South Korea) capable of potential difference up to 150 V. This potential difference of 150 V was applied as pulsed signal of 200 ms generated with LabView software (National Instruments, Austin, TX, USA). Generated droplets move to the second part of the microextraction device which has another inlet for the injection of the TBABrich phase containing ruthenium red (MW 786.36, >85%, Fluka, Buchs, Germany) that has the structural formula of [(NH3 )5 Ru-ORu(NH3 )4 -O-Ru(NH3 )5 ]Cl6 . The produced droplets are swept into the new stream by the shear caused by velocity difference and the microextraction begins in the new stream. The microextrac-
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Fig. 2. Correlation between the brightness from the digital images of ammonium sulfate-rich droplets and ruthenium red concentrations. The digital images in the box on the right show the difference in brightness of the ammonium sulfate-rich phase droplets whose ruthenium red concentrations are 0%, 0.1%, 0.4%, 0.7%, 1.0% and 1.3% (w/w). The expression for the straight line obtained by linear regression is: (concentration) = − 0.013 × (brightness) + 2.37. R2 = 0.9871.
tion device for microextraction control has two branched channels at the position of 10 and 20 mm from the junction of the ruthenium red injection where microextraction begins as shown in Fig. 1b. Microextraction continues until the pulse signal is applied at one of the outlet branches and the droplets are moved into switched to that branch. The concentration of ruthenium red inside the ammonium sulfate-rich droplet at every 10 mm was measured by the analysis of brightness data from digital images. Using the normalized brightness data of the digital images collected by Scion Image software (Scion, Frederick, MD, USA), the concentration of ruthenium red extracted into the ammonium sulfate-rich droplets was analyzed by Photoshop 7 (Adobe, San Jose, CA, USA), after calibrating the data earned from microscopic images of ammonium sulfaterich droplets with known concentrations of ruthenium red. As shown in Fig. 2, brightness data were normalized based on the six ammonium sulfate-rich phase droplets of which concentrations of ruthenium red are 0, 0.1%, 0.4%, 0.7%, 1.0% and 1.3% (w/w), respectively, and the correlation between concentration of ruthenium red and brightness was constructed. Although this is not a direct concentration measurement by spectroscopy, it allows the acquisition of concentration data in situ in the microfluidic device during microextraction. It can avoid any further mass transfer between the droplet and the continuous phase, which is possible in the case of direct sampling of droplets. We collected the brightness data, not from the whole droplet, but within the circle whose radius is 0.7R (R denotes the radius of the whole droplet) not to include the edge rim of the droplet because brightness may not be correct due to the presence of interface. To determine the concentration, the collected brightness information was compared with the baseline brightness value that is the integrated brightness data in the PDMS wall area of the digital image. The data were averaged with at least 10 droplet images that were taken at the same time of the microextraction process. 3. Results and discussion 3.1. Droplet generation A droplet is electrohydrodynamically generated at the ‘dropletgeneration’ part of each device in Fig. 1a and b. TBAB-rich phase and ammonium sulfate-rich phase are introduced individually through the inlets and contacted with each other at the Y-junction, forming laminar flow along the main microchannel. At the T-junction, entire ammonium sulfate-rich phase with smaller viscosity and a
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portion of TBAB-rich phase flow together through one branch of the channel, while the other branched channel at the T-junction is filled with TBAB-rich phase only. When a pulse of d.c. electric potential is applied between the electrodes located at the end of the branched channels, ammonium sulfate-rich phase is attracted toward the positive electrode and the interface is deformed drastically, to dispense a small volume of ammonium sulfate-rich phase as a droplet dispersed in TBAB-rich phase. Details of the droplet generation and its mechanism can be found in our previous reports [12,13]. For droplet generation, several different EHD forces are thought to be acting in combination. While inertia of streams may suppress the generation of droplets, the electrostatic attraction of ammonium sulfate-rich phase toward the positive electrode is the primary force that deforms the interface and generates droplets. The interfacial tension also helps the generation of ammonium sulfate-rich droplets, minimizing the interfacial energy. Once the ammonium sulfate-rich phase stream is elongated by the electric field, the shear force of the TBAB-rich phase stream chops the ammonium sulfaterich phase stream into droplets. The perpendicular channel wall at the T-junction is also essential as a ‘chopping board’ for splitting the extended ammonium sulfate-rich phase stream into droplets. In our experiments, higher voltage is required to generate even a single droplet, when the angle between the main microchannel and branched channel connected to the positive electrode is larger than a right angle. Although the TBAB-rich phase is more hydrophobic than ammonium sulfate-rich phase so that the former phase had constant contact with the PDMS surface which is hydrophobic as well, both phases are basically hydrophilic due to the high water content. In addition, since the TBAB-rich phase is the continuous phase in which ammonium sulfate-rich phase is dispersed, the more hydrophilic nature of the ammonium sulfate-rich phase did not have great effects on the droplet movement in our experiment. The mean radius of the droplets in the experiment was 58.4 m and the standard deviation was 3.8 m. 3.2. Microextraction kinetics To understand microextraction kinetics of ATPS, we employed a microchannel of the length of 70 mm in the extraction part shown in Fig. 1a. This length is sufficient to allow microextraction to continue until color change in droplets is no longer noticed by microscopic observation. The feed flow rates of ammonium sulfate-rich phase and TBAB-rich phase into droplet-generation part are kept at 5.04 l/min and 0.14 l/min, respectively. In the microextraction part, ruthenium red-containing TBAB-rich phase is introduced to the main microextraction channel, as shown in Fig. 1a and b schematically, at the flow rate of 0.24 l/min. The flow rate combination is selected carefully to have appropriate pressure drop and width of each stream, especially at the T-junction in droplet-generation part and at the junction where ruthenium red stream is introduced in microextraction part. Six kinds of ruthenium red-containing TBAB-rich phases were prepared by dissolving ruthenium red in the TBAB-rich phase with the concentration of 0.05%, 0.07%, 0.09%, 0.11%, 0.13% and 0.15% (w/w). Ruthenium red was dispersed in the TBAB-rich phase as solid particles, but was highly soluble in ammonium sulfate-rich phase as identified by its color. We assume the whole extraction process to be composed of two different steps that are clearly observed with optical microscope. One is the process where ruthenium red in the continuous phase is transported to the disperse phase and dissolved on the surface of the ammonium sulfate-rich phase droplet until it is saturated. The general equation for the process involving dissolution of dispersed solid particles can be written as [29,30] ∂Cs = kdiss (Csat − Cs ) ∂t
(1)
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where Cs denotes the concentration at the surface of the droplet, Csat , the concentration when saturated, and kdiss , the dissolution rate constant. Integrating above equation gives Cs as a function of time as follows: Cs = Csat (1 − exp(−kdiss t))
(2)
The second step is the transfer of ruthenium red from the surface of droplet to the center of the droplet. For this step, onedimensional model of simple diffusion with spherical coordinate system is ∂Cin 1 ∂ =D 2 ∂t r ∂r
r
2 ∂Cin
(3)
Fig. 3. Comparison of the experimental (symbols) and simulated data (lines) of the droplet-based microextraction kinetics.
where Cin and D denote the concentration of solute inside the droplet and the effective diffusion coefficient, respectively. This model assumes no internal motion of the fluid in the droplet. The concentration in the continuous phase is assumed to be constant because TBAB-rich phase with fixed concentration of ruthenium red is continuously fed to the microextraction device. The boundary conditions at r = 0 and r = R for this equation are specified as
are shown in Fig. 3. Here, Cout is the concentration of ruthenium red in the continuous TBAB-rich phase. Time-dependent simulation results are in good agreement with the experimental results except the lowest concentration results (Cout = 0.05% (w/w)) in their initial time stage where simulation overestimates the concentrations. One explanation for this error may be that in the early stages of microextraction, the ruthenium red is mainly in the region near the surface of the droplet which is excluded in the brightness measurement especially when the concentration of ruthenium red is very low. As the extraction process continues, the more ruthenium red is diffused to the region belonging to the brightness measurement, and simulation shows better agreement with experimental result. Fig. 4 shows the ratio of the concentration of ruthenium red in the droplet after 45 s of microextraction to that of continuous TBABrich phase fed initially. In the first three cases of low ruthenium red concentrations (0.05%, 0.07% and 0.09% (w/w)) in TBAB-rich phase, the ratio is almost constant. However, the ratio decreases, as the concentration of ruthenium red of the TBAB-rich phase increases for high range of concentration of ruthenium red. This suggests that there exists the solubility limit of ruthenium red in the ammonium sulfate-rich droplet above which the concentration cannot increase independent of the concentration in the TBAB-rich phase. The solubility limit of ruthenium red in the ammonium sulfate-rich phase droplet, corresponding to Cout of 0.13% and 0.15% (w/w), is about 1.15% (w/w) in this experiment.
∂r
,
∂Cin = 0 at r = 0, ∂r
(4)
Cin = Cs = Csat (1 − exp(−kdiss t))
at r = R.
(5)
The initial condition is Cin = 0 at t = 0
for all r.
(6)
The first boundary condition (Eq. (4)) assumes symmetry at the center of the droplet. In the second boundary condition (Eq. (5)), Csat is the concentration on the surface of the droplet at saturation, which corresponds to the product of concentration of ruthenium red in TBAB-rich phase and distribution coefficient between two phases. Although concentration in the TBAB-rich phase is known and assumed to be constant, the distribution coefficient is not available. After sufficiently long extraction time, the droplet will be saturated with solute and the concentration will correspond to Csat . In this work, we assume the extraction time is long enough and the final concentration at the end of extraction is considered to be Csat . Solving the equation numerically with the boundary conditions gives radial distribution of concentration in the droplet at each time of concentration measurement. The average concentration in a spherical region with a radius of 0.7R inside a droplet is obtained by
2 0.7R
Cavg =
0
0
o
Cr 2 sin drdd
2 0.7R 0
0
o
r 2 sin drdd
.
3.3. Microextraction control For the facility in the operation of the process, the extraction time is shown to be controlled by guiding the droplet movement with EHD force. The time-dependent profile of microextraction
(7)
In this equation, interval of integration for r is 0 ≤ r ≤ 0.7R to be consistent with the experimental measurement as explained in the experimental section. To solve the equation numerically, we need information about Csat , kdiss and D. The measured concentration of ruthenium red at 70 mm position which is the end of microchannel was chosen as Csat in each experiment. The kdiss value was determined as 0.09 (s−1 ) by fitting experimental results from the entire microextraction process and applied to solve the equation with five different Csat . (We have used the same Csat for experiments of 0.13% and 0.15% (w/w) assuming that the Csat has reached its solubility limit. The increase of the ruthenium red concentration in TBAB-rich phase from 0.13% to 0.15% did not cause further increase in the concentration in the droplet. So, we assumed that the concentration of 1.15% might be the final concentration which is the solubility limit.) Diffusion coefficient of ruthenium red in ammonium sulfate-rich phase, D, was measured by using ‘T-sensor’ developed by Kamholz et al. [31]. Solutions of simple diffusion model with experimental results
Fig. 4. Ratio of the ruthenium red concentration in the droplet after 45 s of microextraction to that of continuous TBAB-rich phase initially introduced to the microextraction device. The lines indicate that the concentration could reach the solubility limit, not increasing continuously.
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Fig. 5. Microextraction control and manipulation of droplet within the microextraction system shown in Fig. 1b. (a) Beginning of microextraction: The ammonium sulfaterich phase droplet enters the ruthenium red-containing TBAB-rich phase at the bottleneck of the microchannel and microextraction starts. (b) The first branched channel at the position of 10 mm from the starting point of microextraction. In this case, the electric pulse is not applied so that the droplet keeps moving through the microextraction channel. (c) The second branched channel at the position of 20 mm from the starting point of microextraction. Applied electric pulse changes the direction of movement of the droplet toward the branched channel so that the microextraction is terminated and the droplet is guided to other part of the microfluidic system.
process obtained from the experimental and simulation results in Section 3.2, together with carefully designed microextraction device and droplet-motion guiding technique, allow us to determine the time needed for a droplet to reach a certain concentration which can be utilized for the specific research or analysis. The microextraction device for this experiment has two branched channels at the position of 10 and 20 mm from the junction of the ruthenium red injection where microextraction begins as shown in Fig. 1b. The branched channels were arranged to have an angle of 45◦ so that droplets can readily move toward the branched channels. Ammonium sulfate-rich phase that forms laminar flow with TBAB-rich phase in the droplet-generation part of the device is fed at the flow rate 5.4 l/min. The droplet produced in the dropletgeneration part of the device moves to microextraction part, and then extraction starts at the junction where TBAB-rich phase with ruthenium red concentration of 0.29% (w/w) is injected. Ruthenium red favors the ammonium sulfate-rich phase in bulk equilibrium state when the two phases are in contact. Only when the droplet crosses the streamlines and is fully surrounded by the ruthenium red-containing TBAB-rich phase, isotropy of microextraction is guaranteed. To force droplet to enter the ruthenium red stream, we fabricated the microchannel to have a narrow cross-section as shown in Fig. 5a. Because the flow rate of the main TBAB-rich phase, which initially carries the ammo-
nium sulfate-rich droplet, is kept at 0.24 l/min, smaller than that of ruthenium red solution (0.51 l/min), the droplet moves toward the ruthenium red solution due to the pressure difference. Microextraction continues until the d.c. electric pulse with the duration of 200 ms is applied at the end of the branched channel. At that moment the droplet departs ruthenium red-containing TBAB-rich phase by approaching the electrode located at each end of the branched microchannel and microextraction is terminated. As shown in Fig. 5b and c, the thresholds located at 10 and 20 mm positions serve as leaping boards for the droplet. When d.c. electric potential is not applied, the droplet moves freely following its streamline over the threshold as shown in Fig. 5b. When electric potential is applied on the electrode at the end of the branched channel, the droplet changes its direction at the junction so that it leaves the flow stream of ruthenium red-containing TBAB-rich phase and jumps into the branched channel with increased speed due to the electrophoretic effect as shown in Fig. 5c. The ruthenium red concentrations of droplets at the first and second branched microchannel when microextraction is terminated are 0.12% and 0.24% (w/w) and corresponding concentrations which have been predicted by simulation are 0.11% and 0.28% (w/w), which shows that the droplet-based microextraction can be terminated depending on the needs in a process, even before reaching the equilibrium state.
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4. Conclusions
References
Droplet-based microextraction with liquid–liquid ATPS has been investigated in this work. The microfluidic devices for EHD generation of aqueous two-phase droplet and microextraction were integrated into a single system which includes simple d.c. electric potential application and programmed control of droplet movement. Solving the equations of simple mass balance and one-dimensional diffusion in spherical coordinate for each step of the microextraction process with appropriate boundary conditions gave solutions that are in good agreement with the experimental data. For the facility in operation, the technique to stop microextraction process was demonstrated by changing the droplet movement direction based on the time-dependent microextraction profile that was investigated by the experiments. We have also tried to predict the concentration by the numerical solution of the simple model of the two-step molecular transport involving diffusion and dissolution. These results suggest that a single microfluidic system can be designed and fabricated to produce droplets with desired solute concentrations. Droplets of known concentrations of certain solute can be further used in the same microfluidic system for analysis, reaction and other purposes if the solute is preferentially distributed in the dispersed phase of the ATPS. From our experiences, the solutes can be inorganic particles, metal ions, and chelating agents. A wider range of application of ATPS droplet-based microfluidic system reported in this paper is expected to take the full advantage of the benefits that the system can provide.
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Acknowledgement This work was supported by Center for Ultramicrochemical Process Systems sponsored by Ministry of Education, Science and Technology.