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Design and modeling of microfluidic systems for multiple chromatographic separations
Microchemical Journal 123 (2015) 125–130
Contents lists available at ScienceDirect
Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Design and modeling of microfluidic systems for multiple chromatographic separations Andrea Nagy a, Eszter L. Tóth b, Kristóf Iván b, Attila Gáspár a,⁎ a b
Department of Inorganic and Analytical Chemistry, University of Debrecen, Debrecen, Hungary Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest, Hungary
a r t i c l e
i n f o
Article history: Received 17 April 2015 Accepted 21 May 2015 Available online 28 May 2015 Keywords: Microchip Computational fluid dynamic simulation Chromatography Polydimethylsiloxane
a b s t r a c t In this work the design of a microchannel system applied for multiple chromatographic separations was optimized. The pressure and velocity distribution in a complex microchip was simulated by COMSOL Multiphysics software in order to find the optimized geometry and pressure conditions. The experimental results of the flow rates in the twelve parallel channels of the used microchip agreed well with the simulation results. COMSOL simulations were also applied to find adequate channel designs for the equalization of the flow rates in the parallel channels of a chip, which included many junctions of channels and bottlenecks. This approach can greatly expedite the time required for complex geometry based prototype fabrication. © 2015 Published by Elsevier B.V.
1. Introduction Microfluidic chips have attracted great attention as they offer several advantages over conventional analytical techniques including small sample/reagent volume requirements, portability, ability to multiplex and compatibility with other techniques [1]. Also, there is a high demand for miniaturized chromatographic techniques that would provide the same very versatile applications that have been found in liquid chromatography (LC) [2]. Recently, Hansen et al. [3,4] reported high-quality microfluidic solid-phase chromatography columns formed by multilayer soft lithography and an application of special bypass channel systems along the length of the chromatographic packing. In the same year, we reported that even a temporary tapering of the channel in a microchip made of soft PDMS can lead to effective retaining of the chromatographic particles [5]. For the parallel multi-channel chromatographic separations several channel patterns were designed. The obtained multipackings were applied for parallel separation of dyes [5]. The implementation of chromatographic separation units on the microscale allows faster and high throughput separations. One of the most advantageous features of microfluidic systems that complex channel designs can be easily fabricated, and often through the multiplication of the channel systems the overall complexity of the system further increases. In such a complex channel system, where several junctions of the channels split or merge streams of the liquid, the flow rates and pressure conditions are not easy to estimate. However, in many applications of microchips the flow rates in certain parts of
⁎ Corresponding author. Tel.: +36 30 2792889; fax: +36 52 518660. E-mail address: [email protected] (A. Gáspár).
http://dx.doi.org/10.1016/j.microc.2015.05.019 0026-265X/© 2015 Published by Elsevier B.V.
the channels should be well established, or controlled by changing the geometry of the channel design [1,2]. COMSOL Multiphysics® is sophisticated simulation software for various engineering applications, which offers an excellent and convenient way to optimize the microfluidic channel design prior to the microfabrication of the prototype, thus it significantly reduces the time required for testing, optimizing and fabrication [6]. COMSOL was successfully applied for the simulation of microfluidic systems, optimization of physical parameters (eg. impedance modulation of a nanoneedle [7], acoustic streaming and radiation forces [8]). Toepke et al. determined the velocity and concentration profiles in a microfluidic flow-flash system [9]. Others used COMSOL simulations to fine-tune the channel parameters and to validate the designed channel pattern [10]. Haroun et al. made simulations to find the ideal channel pattern providing the efficient synthesis of a PET radiotracer compound [11]. In the present work COMSOL was used to simulate the flow rate distribution in a channel system, which included several junctions of channels and bottlenecks (microchip with injectors and 12 chromatographic packings). 2. Experimental 2.1. Design and fabrication of the microchip Microfluidic chips made of polydimethylsiloxane (PDMS) were prepared by using a mold replica created by soft photolithography mainly according to the procedure described by Whitesides et al. [12]. The channel layout was printed as a high-resolution (4000 dpi) photomask. In order to get a thickness of around 40 μm the negative type photoresist (SU-8 2025, Microchem, Newton, MA) was spin-coated onto a
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3″ silicon wafer with 2500 rpm for 60 s. The PDMS chip was fabricated by a cast molding of a 10:1 mixture of PDMS oligomer and crosslinking agent (Sylgard 184, Dow Corning, Midland, MI). The PDMS chip was sealed onto a 1.2 mm thick glass slide after air plasma treatment (PDC-32G, Harrick, Ithaca, NY). The microchip includes 12 parallel 100 μm wide channels, which merge into a single port O3 of a 0.3 mm diameter (Fig. 1.a and 1.b). At the other end of the channel system, similar ports (port I for liquid/ sample inlet for separation, ports O1, O2, O4 for waste outlets) are created by punching the PDMS. For the preparation of chromatographic packing, similar process was applied that was used in our earlier works [13,14]. The large number (eg.: 12) of channels could be packed simultaneously from the single common exit port O3, pumping a single plug of suspension of C18 chromatographic particles (Varian, Inc. Walnut Creek, CA, USA) toward the parallel bottlenecks (Fig. 1.a and 1.b). The packing was prepared daily, and before the chromatographic separation/preconcentration the packings were conditioned with 10 min washing with methanol and 5 min with the mobile phase (water). 2.2. Parallel chromatographic separations in the microchip The formation of around 1 nL sample plug in a crossing of channels just before the chromatographic packing was a consequence of the high hydraulic resistance of the packing, which, in turn, resulted in a
reduced flow rate in the separation channel. When a sample plug introduced from port I reaches the junction of channels, the sample flows into three other channels of the intersection with different flow rates depending on the hydraulic resistance of each channel (Fig. 1.b). This split-flow pressure injection is described in our earlier works [13, 15]. The hydraulic resistance in the separation channel of chip is estimated to be approximately one thousand times higher (i.e. the flow rate is one thousand times smaller) than in the channel at the inlet port I. Therefore, when 1 μL of sample is injected into the chip with a peristaltic pump, only about 1 nL is injected into the separation channels (toward port O3), that is, the majority of the sample solution flows to the waste outlet reservoirs O1 and O2. To observe the movement of liquids in the microchip channels and to test the chromatographic characteristics of the packing, food dyes (FD&C blue#1 and FD&C yellow#5, all from McCormick&Co., Inc., Sparks, MD, USA) were injected and transported in the chip using a low-rate peristaltic pump (IPC, Ismatec). While in aqueous solution the blue dye was completely retained, the yellow dye passed through the packing (Fig. 1.c). Changing the mobile phase to methanol, the blue component was quickly washed out (Fig. 1.d) (Video-1). Thus the two components could be separated within 30 s. The flow rates in the parallel channels of the microchip of Fig. 1 gradually increase from one side to the other side (the flow rate in the outermost left (1st) channel was around 2.5 times larger than in the
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Fig. 1. Multiple chromatographic separations on microchip. Photographic (a) and schematic (b) representation of the microchip with 12 parallel C18 packings (0.1 mm × 0.03 mm × 10 mm, as width × height × length). In the left top corner of (a) the parallel channels packed with the chromatographic particles are shown with dark background. The peristaltic pump tubing is connected to port I and the other outlets (port O1, O2, O3 and O4). Optical micrographs of the parallel C18 packings on the microchip for separation of blue and yellow dye. A single sample (1 μL) was injected to the chip through port I and it was split to several equal parts before the packings. While the blue dye is completely retained at the beginning of the packing, the yellow dye was washed out by mobile water phase (c). Changing the mobile phase to methanol the blue dye was eluted (d). The flow velocities in the parallel channels of the microchip were measured based on the video of the chromatographic elutions of the blue dye through the parallel packings (e).
A. Nagy et al. / Microchemical Journal 123 (2015) 125–130
outermost right (12th) channel) [5]. The flow velocities in the parallel channels were increasing in a linear manner (Fig. 1.e). 2.3. Flow visualization and measurement of the velocities of fluid flow in the chip The movement of the plugs of the dyes was monitored by an inverted microscope (Axio ObserverA1, Zeiss) equipped with a high speed CCD camera. Videos and the images were recorded and the color (RGB) intensities were measured by AxioVision 4.6.3 (Zeiss) software. The separated bands were detected by the microscope camera recording the RGB intensities at a specified part of the channel (eg. at the end of the packing). Since the transparent PDMS chip includes thin micropackings of the transparent silica particles, the movement of the bands of the dyes can be monitored within the packings as well. This gives a convenient way to make a chromatogram about the separation of dyes recorded as a video (Video-1). The velocities of the fluid flow in the different part of the chip could be determined from the video stream. 2.4. Computational fluid dynamics simulations In our work computational fluid dynamics was used to simulate and predict the flow rates in a system of microchannels using COMSOL Multiphysics 5.0 version (COMSOL, Inc., Palo Alto, CA, USA). COMSOL Multiphysics is a Finite Element Method (FEM) based on simulation software for various engineering applications. The Computational Fluid Dynamics (CFD) module of the program was expected to be well-applicable for microfluidic chips, too. For the simulation laminar and incompressible fluid flow was assumed and the Navier–Stokes equation (Eq. 1) was calculated numerically:
∂v 1 þ ðv ∇Þv ¼ − ∇p þ ν∇2 v; ρ ∂t
ð1Þ
where v is the velocity (m/s), p is pressure (Pa), ρ is density (kg/m3) and ν is the kinematic viscosity (m2/s) of the fluid. Laminar inflow boundary condition was applied at the inlet with laminar profile and 10 μL min−1 flow rate (in accordance with the flow rate generated by the peristaltic pump) and zero pressure boundary condition was applied at the outlet. For the channel walls no slip boundary condition was set. Fluid properties were in accordance with the parameters of the room temperature water (density: 1000 kg m−3, kinematic viscosity: 10−6 m2 s−1). The velocity profiles and pressure distribution of the parallel channels were calculated in 2D model using triangular mesh. The number of mesh elements varied around 40 000 with an average element size of 25 μm and a minimum element size of 3 μm corresponding number of degrees of freedom up to 105. The results were obtained graphically as pressure and velocity distribution images. Numeric values were also exported to analyze the velocity and pressure profiles along lines. 3. Results and discussion Although in microfluidic systems very complex channel designs can be easily fabricated, due to the complexity of several splitting/merging junctions of the channels and the different geometries (height, width, shape) of the channel design, the flow rates and pressure conditions are difficult or impossible to estimate without numeric calculations. Questions that can be verified through simulations are typically like these: • What is the flow direction at a certain part of the chip? • What is the ratio of flow rates in the different channels of the chip?
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• How is a given volume of sample plug distributed between the channels after passing a junction? • What are the flow profiles (velocity gradient) like in the cross-section of a channel?
Since the Reynolds number in the microfluidic channels is very small (generally less than 1), the fluid flow in a pressure driven system should be considered as strongly laminar. The laminar flow allows analytes (a portion of liquid) to be transported in a predictable way in the microchip. Laminar flow simplifies the numeric simulation, as well because it does not require the multiscale approximation of turbulence. When chromatographic separations performed in parallel or sequentially are compared, the constancy of the retention times of the analytes, that is, the constant flow rates in the chromatographic packings are crucially important. Complex microfluidic channel systems (with several junctions and varying channel dimensions, ports, bottlenecks, etc.) for multiple chromatographic separations are relatively simple to create with microfabrication, but when the channel pattern design is ready the flow rates through the multiple chromatographic packings are often difficult to estimate. The fluid velocities in the parallel channels of the microchip shown in Fig. 1 gradually increase from one side to the other side and the ratio of the maximal and minimal velocity in the parallel channels was around 2.5 [5]. The flow rate and pressure field in the microchip described in Fig. 1 were simulated by COMSOL. The liquid (water) was pumped into the microchip through the inlet port I, where the flow rate was adjusted to 10 μL min− 1 (around 60 Pa) and the liquid could leave the chip through the ports O1–O4, where the pressure was fixed at 0 Pa. The small volume of sample plug was formed after the junction of the channels (Fig. 2.b and 2.c), because there the flow rate was only 0.32 μL min− 1, while the flow rates toward ports O1 and O2 were 9.57 μL min− 1. The flow rate toward the parallel channels (other outlets) is much smaller due to the hydrodynamic resistance caused by the much longer channel and bottlenecks. The simulation was calculated for empty channels because the back-pressure of the chromatographic packings is difficult to estimate by flow simulation. The backpressure of the chromatographic packings largely decreases the flow rate toward the parallel packings (and thus the volume of the sample plug). In Fig. 2.d and 2.e the pressure distribution and velocity magnitude can be examined in the complete channel system of the microchip. From these diagrams the relatively large differences in the flow velocities and the pressure between the different channels can be concluded. The differences between the flow rates can be illustrated also by plotting a line section (marked as dotted line in Fig. 2.a) perpendicular to the parallel channels (Fig. 2.f). As it was expected from the experimental observations (Video-1) the flow rates increased from one side to the other side, but the ratio of the maximal and minimal velocities (vmax/vmin) simulated in the parallel channels was amount to 3.4, slightly higher than in the experiments, which was measured to be 2.5. To understand what set of parameters could lead to this difference, the conditions of the transportation of the liquid in the chip were changed for the COMSOL simulations. The ports of the PDMS chips are created by punching the soft material. The dimension and the surface of the punched vertical channel and the obtained orifice at the end of the channel are rather occasional in microscopic scale. Smaller or larger bottlenecks can often form in the ports, which result in a small back-pressure against the liquid flow driven from port I. In a series of simulation experiments the values of the back-pressure at the port O4 were adjusted to 0 Pa, 0.68 Pa, 1 Pa, 6.9 Pa and 10 Pa. Increasing the back-pressure at port O4 the flow velocity difference of the parallel channels decreased (Fig. 3). However, after an application of back-pressure of around 2 Pa, instead of a monotonous increase of the flow rates from the left channels to right ones, the flow rates were the lowest in the middle channels and highest in the sidechannels (Fig. 3). Using 6.9 Pa back-pressure the flow rates in the two
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Fig. 2. COMSOL simulations of pressure distribution (b) and flow rates (c) in the sample introduction part (junction of fluid channels) and in the complete channel system (d, and e,) of the microchip. The flow rate applied at the inlet port I was 10 μL min−1, and the pressure at the outlet ports O1–O4 were set to 0 Pa. The line graph of the velocity profiles plotted in the points of a straight line (marked as dotted line in (a)) crossing each parallel channel perpendicularly (f).
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Channels Fig. 3. Maximum velocities along the centerline in the parallel channels obtained by COMSOL simulations using different back-pressures (0, 0.68, 1, 6.9, 10 Pa) at the outlet port O4. The flow rate applied at the inlet port I was 10 μL min−1, and the pressure at the outlet ports O1–O3 were set to 0 Pa. In case of the application of 6.9 Pa back-pressure at outlet port O4 the line graph is shown. The maximum velocity magnitudes from each parallel channel are calculated from the velocity profiles measured on the centerline (marked as dotted line in (Fig. 2.a)).
outermost (left and right) channels were equal, however the ratio of the highest and lowest rate was 1.2, which was found to be the best condition to reach similar flow rates. Increasing the back-pressure further, the flow rates become higher in the left-side channels (which are located closer to port O4). We suppose that the back-pressure in a range of 0– 1 Pa might be formed by the “non-perfect” dimension/shape of the outlet port. Although this pressure is less than 1% of the pumping pressure in the inlet port I, it has considerable effect on the distribution of the flow rate in the parallel channels. When the applied pressure at port O4 was changed from 0 Pa to 1 Pa with 0.01 Pa steps, the ratio between the maximum and minimum velocities was calculated. In this study it was found that the application of 0.68 Pa resulted in the same distribution of flow rates (when the ratio of the maximal and minimal flow rates was 2.5) that was experimentally obtained. The closing the port O4 did not help equalize the flow rates in the parallel channels, the vmax/vmin was found to be 1.9. If the O4 port is set as inlet (I2) with 10 μL min−1 flow rate similar to the original inlet (I1), the flow velocities gradually reduced from the left side to the right side and the vmax/vmin was found to 1.9. From these results it can be concluded that the presence of a small (just a few Pa) back-pressure can have a quite large effect on the velocity distribution. A small back-pressure can be generated by the difference of liquid heights (hydrostatic pressure) or bottlenecks in the ports. Because of the reproducible generation of a small (0–10 Pa), constant pressure in practical microfluidics is difficult, the adjustment of the back-pressure is not a proper way of equalizing the flow rates in the parallel channels. Pumping the liquid into the microchip through port I with different flow rates as 0.1 μL min−1, 1 μL min−1, 10 μL min−1 and 100 μL min−1 has practically no effect on the distribution of flow rates, the vmax/vmin values were 3.37, 3.37, 3.40, and 3.43, respectively. Although the Reynolds number increases proportionally with the increasing flow rate, the largest Reynolds number is only 0.2 (for the 100 μL min−1 pumping rate) showing the strong laminarity of the system. The flow profiles in the channels flatten with the reduction of the flow rate. Additionally, the equalization of the flow rates in the parallel channels was tried to achieve by the modification of the channel geometry. The shape of the straight-line channel before the bottlenecks was
A. Nagy et al. / Microchemical Journal 123 (2015) 125–130
converted to a trapezoid shape one, where the ratio of the opposite parallel sides d1/d2 was set to 1:1, 2:1, 4:1, 8:1 (Fig. 4.a). Due to this trapezoid shape of the channel, the flow in front of the bottlenecks slowed down close to the longer side of the trapezoid (d1). When the ratio of the d1/d2 was changed to 2:1, the vmax/vmin value was improved (decreased) to 1.65, but if the d1/d2 was set to 8, the flow rates were equalized (vmax/vmin = 1.0) (Fig. 4.b). The velocity and pressure field diagrams for the microchip, where the ratio of the d1/d2 was 8, are shown in Fig. 4.c and 4.d. From the diagram of Fig. 4.c it can be concluded that at the acute angle of the trapezoid channel the flow rate of the liquid is almost zero, that is, the dead-volume of the trapezoid channel can be partly reduced by making it round as it was shown in Fig. 4.e. The previous simulations were made for the microchips that include exclusively open channels. When the parallel channels are homogeneously packed with chromatographic packing materials, the flow rates will be much smaller due to the high back-pressure, however the velocity distribution remains the same as in open channels. The microchip having multiple channels with equalized flow rates as the design optimized by COMSOL simulation was created with microfabrication and the parallel channels were uniformly packed with C18 modified silica particles of 5 mm (Fig. 5.a). Repeating the chromatographic separations of the two dyes in this microchip as it was shown in Fig. 1 and Video-1, now the flow rates in the 12 channels became very similar, the vmax/vmin equals to 1.10 (Fig. 5.d) (Video-2). The distribution of the flow rates can be simply determined based on the movement of both the firstly eluted yellow dye (Fig. 5.b) and the later eluted blue dye profiles (Fig. 5.c). In the microchips with 12 parallel chromatographic packings shown in Figs. 1–5 only one sample could be injected, which was then split
a,
to 12 equals parts before the bottlenecks. Since a large number of parallel and independent analytical systems can be arranged on a single microchip, the implementation of multiple chromatographic separations for several samples at the same time is probably also possible. Currently, a three-layer microchip design, which makes it possible to separately transfer samples from each sample reservoirs is being studied with the use of simulations. 4. Conclusions In microfluidic devices, especially if those include complex channel system, alterations between the experimentally gained and the expected (desired) flow rates at given parts in the microchip are often obtained. Without the use of simulation software it is quite difficult to get the channel pattern with the right dimensions or the best experimental parameters (flow rates, pumping pressure). Using simulation the exhausting preparation of microfluidic chip (including lithographic masks, molds) with trial and error method can be avoided. It was shown that COMSOL can help to better understand why the velocity distribution obtained in a microchip sometimes differs from that we expected. Our results also prove that a channel design optimized by COMSOL produces the same performance and characteristics as the experimental microfluidic chip fabricated in a laboratory. We designed a chip, in the parallel channels of which uniform velocities of the flow could be achieved, which is essential to provide for chromatographic separations. We studied the effect of opening or closing certain ends of the channels, application of pressure/back-pressure at several parts of the chip, but the best result was achieved with the modification of dimensions (shape) of the channel pattern. The COMSOL simulations matched well with the obtained experimental results.
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Fig. 4. Studies for equalization of the flow rates in the parallel channels by the modification of the channel pattern. The schematic drawing of the microchip, where the ratios of the d1/d2 (widths of the channel before the bottlenecks) were changed between 1 and 8 (a). The results of the simulation: velocity distribution in the channels using channel patterns with different ratios of the d1/d2 (b), velocity (c) and pressure distribution (d) diagrams for microchip where the ratio of the d1/d2 was 8.
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Fig. 5. Multiple chromatographic separations on microchip. The schematic representation (a) of the microchip with 12 parallel C18 packings which is identical to the one described in Fig. 1 except the channel before the bottlenecks has a trapezoid shape, where the ratio of the opposite parallel sides d1/d2 were set to 8:1. While the blue dye was completely retained at the beginning of the packing, the yellow dye was washed out by mobile water phase (b). Changing the mobile phase to methanol the blue dye was eluted (c). The linear flow rates in the parallel channels of the microchip were measured based on the video of the chromatographic elutions of the blue dye through the parallel packings (d).
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.microc.2015.05.019. Acknowledgments The authors gratefully acknowledge the financial support for this research by grants from the National Scientific Research Fund, Hungary (OTKA K75286), the EU and co-financed by the European Social Fund under the project SROP-4.2.2.B-15/1/KONV-2015-0001, the Pázmány University grant KAP-1.1-14/024 (recipient: Kristóf Iván) and the Hungarian NAP grant no.: KTIA-NAP_13-1-2013-0001. References [1] P. Tabeling (Ed.), Introduction to Microfluidics, University Press, Oxford, 2005. [2] J.P. Kutter, Liquid phase chromatography on microchips, J. Chromatogr. A 1221 (2012) 72–82. [3] J. Huft, C.A. Haynes, C.L. Hansen, Fabrication of high-quality microfluidic solid-phase chromatography columns, Anal. Chem. 85 (2013) 1797–1802. [4] J. Huft, C.A. Haynes, C.L. Hansen, Microfluidic integration of parallel solid-phase liquid chromatography, Anal. Chem. 85 (2013) 2999–3005. [5] A. Nagy, A. Gaspar, Packed multi-channels for parallel chromatographic separations in microchips, J. Chromatogr. A 1304 (2013) 251–256. [6] S. Cohin (Ed.), Microfluidics, Wiley, Hoboken, USA, 2010.
[7] R. Esfandyarpour, H. Esfandyarpour, J.S. Harris, R.W. Davis, Simulation and fabrication of a new novel 3D injectable biosensor for high throughput genomics and proteomics in a lab-on-a-chip device, Nanotechnology 24 (2013) 465301 (p11). [8] S.M. Hasgsäter, T. Glasdam Jensen, H. Bruus, J.P. Kutter, Acoustic resonances in microfluidic chips: full-image micro-PIV experiments and numerical simulations, Lab Chip 7 (2007) 1336–1344. [9] M.W. Toepke, S.H. Brewer, D.M. Vu, K.D. Rector, J.E. Morgan, R.B. Gennis, P.J.A. Kenis, R.B. Dyer, Microfluidic flow-flash: method for investigating protein dynamics, Anal. Chem. 79 (2007) 122–128. [10] L. Saias, J. Autebert, L. Malaquin, J-L. Viovy, Design, modeling and characterization of microfluidic architectures for high flow rate, small footprint microfluidic systems, Lab Chip 11 (2011) 822–832. [11] S. Haroun, L. Wang, T.J. Ruth, P.C.H. Li, Computational fluid dynamics study of the synthesis process for a PET radiotracer compound, [11C]raclopride on a microfluidic chip, Chem. Eng. Process. 70 (2013) 140–147. [12] D.C. Duffy, J.C. McDonald, O.J.A. Schueller, G.M. Whitesides, Rapid prototyping of microfluidic systems in poly(dimethylsiloxane), Anal. Chem. 70 (1998) 4974–4984. [13] A. Gaspar, M.E. Piyasena, F.A. Gomez, Fabrication of fritless chromatographic microchips packed with conventional reversed-phase silica particles, Anal. Chem. 79 (2007) 7906–7909. [14] A. Gaspar, A. Nagy, I. Lazar, Integration of ground aerogel particles as chromatographic stationary phase into microchip, J. Chromatogr. A 1218 (2011) 1011–1015. [15] A. Gaspar, P.I. Koczka, H. Carmona, F.A. Gomez, Split injection: a simple introduction of subnanoliter sample volumes for chip electrophoresis, Microchem. J. 99 (2011) 180–185.
Contents lists available at ScienceDirect
Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Design and modeling of microfluidic systems for multiple chromatographic separations Andrea Nagy a, Eszter L. Tóth b, Kristóf Iván b, Attila Gáspár a,⁎ a b
Department of Inorganic and Analytical Chemistry, University of Debrecen, Debrecen, Hungary Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest, Hungary
a r t i c l e
i n f o
Article history: Received 17 April 2015 Accepted 21 May 2015 Available online 28 May 2015 Keywords: Microchip Computational fluid dynamic simulation Chromatography Polydimethylsiloxane
a b s t r a c t In this work the design of a microchannel system applied for multiple chromatographic separations was optimized. The pressure and velocity distribution in a complex microchip was simulated by COMSOL Multiphysics software in order to find the optimized geometry and pressure conditions. The experimental results of the flow rates in the twelve parallel channels of the used microchip agreed well with the simulation results. COMSOL simulations were also applied to find adequate channel designs for the equalization of the flow rates in the parallel channels of a chip, which included many junctions of channels and bottlenecks. This approach can greatly expedite the time required for complex geometry based prototype fabrication. © 2015 Published by Elsevier B.V.
1. Introduction Microfluidic chips have attracted great attention as they offer several advantages over conventional analytical techniques including small sample/reagent volume requirements, portability, ability to multiplex and compatibility with other techniques [1]. Also, there is a high demand for miniaturized chromatographic techniques that would provide the same very versatile applications that have been found in liquid chromatography (LC) [2]. Recently, Hansen et al. [3,4] reported high-quality microfluidic solid-phase chromatography columns formed by multilayer soft lithography and an application of special bypass channel systems along the length of the chromatographic packing. In the same year, we reported that even a temporary tapering of the channel in a microchip made of soft PDMS can lead to effective retaining of the chromatographic particles [5]. For the parallel multi-channel chromatographic separations several channel patterns were designed. The obtained multipackings were applied for parallel separation of dyes [5]. The implementation of chromatographic separation units on the microscale allows faster and high throughput separations. One of the most advantageous features of microfluidic systems that complex channel designs can be easily fabricated, and often through the multiplication of the channel systems the overall complexity of the system further increases. In such a complex channel system, where several junctions of the channels split or merge streams of the liquid, the flow rates and pressure conditions are not easy to estimate. However, in many applications of microchips the flow rates in certain parts of
⁎ Corresponding author. Tel.: +36 30 2792889; fax: +36 52 518660. E-mail address: [email protected] (A. Gáspár).
http://dx.doi.org/10.1016/j.microc.2015.05.019 0026-265X/© 2015 Published by Elsevier B.V.
the channels should be well established, or controlled by changing the geometry of the channel design [1,2]. COMSOL Multiphysics® is sophisticated simulation software for various engineering applications, which offers an excellent and convenient way to optimize the microfluidic channel design prior to the microfabrication of the prototype, thus it significantly reduces the time required for testing, optimizing and fabrication [6]. COMSOL was successfully applied for the simulation of microfluidic systems, optimization of physical parameters (eg. impedance modulation of a nanoneedle [7], acoustic streaming and radiation forces [8]). Toepke et al. determined the velocity and concentration profiles in a microfluidic flow-flash system [9]. Others used COMSOL simulations to fine-tune the channel parameters and to validate the designed channel pattern [10]. Haroun et al. made simulations to find the ideal channel pattern providing the efficient synthesis of a PET radiotracer compound [11]. In the present work COMSOL was used to simulate the flow rate distribution in a channel system, which included several junctions of channels and bottlenecks (microchip with injectors and 12 chromatographic packings). 2. Experimental 2.1. Design and fabrication of the microchip Microfluidic chips made of polydimethylsiloxane (PDMS) were prepared by using a mold replica created by soft photolithography mainly according to the procedure described by Whitesides et al. [12]. The channel layout was printed as a high-resolution (4000 dpi) photomask. In order to get a thickness of around 40 μm the negative type photoresist (SU-8 2025, Microchem, Newton, MA) was spin-coated onto a
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3″ silicon wafer with 2500 rpm for 60 s. The PDMS chip was fabricated by a cast molding of a 10:1 mixture of PDMS oligomer and crosslinking agent (Sylgard 184, Dow Corning, Midland, MI). The PDMS chip was sealed onto a 1.2 mm thick glass slide after air plasma treatment (PDC-32G, Harrick, Ithaca, NY). The microchip includes 12 parallel 100 μm wide channels, which merge into a single port O3 of a 0.3 mm diameter (Fig. 1.a and 1.b). At the other end of the channel system, similar ports (port I for liquid/ sample inlet for separation, ports O1, O2, O4 for waste outlets) are created by punching the PDMS. For the preparation of chromatographic packing, similar process was applied that was used in our earlier works [13,14]. The large number (eg.: 12) of channels could be packed simultaneously from the single common exit port O3, pumping a single plug of suspension of C18 chromatographic particles (Varian, Inc. Walnut Creek, CA, USA) toward the parallel bottlenecks (Fig. 1.a and 1.b). The packing was prepared daily, and before the chromatographic separation/preconcentration the packings were conditioned with 10 min washing with methanol and 5 min with the mobile phase (water). 2.2. Parallel chromatographic separations in the microchip The formation of around 1 nL sample plug in a crossing of channels just before the chromatographic packing was a consequence of the high hydraulic resistance of the packing, which, in turn, resulted in a
reduced flow rate in the separation channel. When a sample plug introduced from port I reaches the junction of channels, the sample flows into three other channels of the intersection with different flow rates depending on the hydraulic resistance of each channel (Fig. 1.b). This split-flow pressure injection is described in our earlier works [13, 15]. The hydraulic resistance in the separation channel of chip is estimated to be approximately one thousand times higher (i.e. the flow rate is one thousand times smaller) than in the channel at the inlet port I. Therefore, when 1 μL of sample is injected into the chip with a peristaltic pump, only about 1 nL is injected into the separation channels (toward port O3), that is, the majority of the sample solution flows to the waste outlet reservoirs O1 and O2. To observe the movement of liquids in the microchip channels and to test the chromatographic characteristics of the packing, food dyes (FD&C blue#1 and FD&C yellow#5, all from McCormick&Co., Inc., Sparks, MD, USA) were injected and transported in the chip using a low-rate peristaltic pump (IPC, Ismatec). While in aqueous solution the blue dye was completely retained, the yellow dye passed through the packing (Fig. 1.c). Changing the mobile phase to methanol, the blue component was quickly washed out (Fig. 1.d) (Video-1). Thus the two components could be separated within 30 s. The flow rates in the parallel channels of the microchip of Fig. 1 gradually increase from one side to the other side (the flow rate in the outermost left (1st) channel was around 2.5 times larger than in the
c,
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Fig. 1. Multiple chromatographic separations on microchip. Photographic (a) and schematic (b) representation of the microchip with 12 parallel C18 packings (0.1 mm × 0.03 mm × 10 mm, as width × height × length). In the left top corner of (a) the parallel channels packed with the chromatographic particles are shown with dark background. The peristaltic pump tubing is connected to port I and the other outlets (port O1, O2, O3 and O4). Optical micrographs of the parallel C18 packings on the microchip for separation of blue and yellow dye. A single sample (1 μL) was injected to the chip through port I and it was split to several equal parts before the packings. While the blue dye is completely retained at the beginning of the packing, the yellow dye was washed out by mobile water phase (c). Changing the mobile phase to methanol the blue dye was eluted (d). The flow velocities in the parallel channels of the microchip were measured based on the video of the chromatographic elutions of the blue dye through the parallel packings (e).
A. Nagy et al. / Microchemical Journal 123 (2015) 125–130
outermost right (12th) channel) [5]. The flow velocities in the parallel channels were increasing in a linear manner (Fig. 1.e). 2.3. Flow visualization and measurement of the velocities of fluid flow in the chip The movement of the plugs of the dyes was monitored by an inverted microscope (Axio ObserverA1, Zeiss) equipped with a high speed CCD camera. Videos and the images were recorded and the color (RGB) intensities were measured by AxioVision 4.6.3 (Zeiss) software. The separated bands were detected by the microscope camera recording the RGB intensities at a specified part of the channel (eg. at the end of the packing). Since the transparent PDMS chip includes thin micropackings of the transparent silica particles, the movement of the bands of the dyes can be monitored within the packings as well. This gives a convenient way to make a chromatogram about the separation of dyes recorded as a video (Video-1). The velocities of the fluid flow in the different part of the chip could be determined from the video stream. 2.4. Computational fluid dynamics simulations In our work computational fluid dynamics was used to simulate and predict the flow rates in a system of microchannels using COMSOL Multiphysics 5.0 version (COMSOL, Inc., Palo Alto, CA, USA). COMSOL Multiphysics is a Finite Element Method (FEM) based on simulation software for various engineering applications. The Computational Fluid Dynamics (CFD) module of the program was expected to be well-applicable for microfluidic chips, too. For the simulation laminar and incompressible fluid flow was assumed and the Navier–Stokes equation (Eq. 1) was calculated numerically:
∂v 1 þ ðv ∇Þv ¼ − ∇p þ ν∇2 v; ρ ∂t
ð1Þ
where v is the velocity (m/s), p is pressure (Pa), ρ is density (kg/m3) and ν is the kinematic viscosity (m2/s) of the fluid. Laminar inflow boundary condition was applied at the inlet with laminar profile and 10 μL min−1 flow rate (in accordance with the flow rate generated by the peristaltic pump) and zero pressure boundary condition was applied at the outlet. For the channel walls no slip boundary condition was set. Fluid properties were in accordance with the parameters of the room temperature water (density: 1000 kg m−3, kinematic viscosity: 10−6 m2 s−1). The velocity profiles and pressure distribution of the parallel channels were calculated in 2D model using triangular mesh. The number of mesh elements varied around 40 000 with an average element size of 25 μm and a minimum element size of 3 μm corresponding number of degrees of freedom up to 105. The results were obtained graphically as pressure and velocity distribution images. Numeric values were also exported to analyze the velocity and pressure profiles along lines. 3. Results and discussion Although in microfluidic systems very complex channel designs can be easily fabricated, due to the complexity of several splitting/merging junctions of the channels and the different geometries (height, width, shape) of the channel design, the flow rates and pressure conditions are difficult or impossible to estimate without numeric calculations. Questions that can be verified through simulations are typically like these: • What is the flow direction at a certain part of the chip? • What is the ratio of flow rates in the different channels of the chip?
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• How is a given volume of sample plug distributed between the channels after passing a junction? • What are the flow profiles (velocity gradient) like in the cross-section of a channel?
Since the Reynolds number in the microfluidic channels is very small (generally less than 1), the fluid flow in a pressure driven system should be considered as strongly laminar. The laminar flow allows analytes (a portion of liquid) to be transported in a predictable way in the microchip. Laminar flow simplifies the numeric simulation, as well because it does not require the multiscale approximation of turbulence. When chromatographic separations performed in parallel or sequentially are compared, the constancy of the retention times of the analytes, that is, the constant flow rates in the chromatographic packings are crucially important. Complex microfluidic channel systems (with several junctions and varying channel dimensions, ports, bottlenecks, etc.) for multiple chromatographic separations are relatively simple to create with microfabrication, but when the channel pattern design is ready the flow rates through the multiple chromatographic packings are often difficult to estimate. The fluid velocities in the parallel channels of the microchip shown in Fig. 1 gradually increase from one side to the other side and the ratio of the maximal and minimal velocity in the parallel channels was around 2.5 [5]. The flow rate and pressure field in the microchip described in Fig. 1 were simulated by COMSOL. The liquid (water) was pumped into the microchip through the inlet port I, where the flow rate was adjusted to 10 μL min− 1 (around 60 Pa) and the liquid could leave the chip through the ports O1–O4, where the pressure was fixed at 0 Pa. The small volume of sample plug was formed after the junction of the channels (Fig. 2.b and 2.c), because there the flow rate was only 0.32 μL min− 1, while the flow rates toward ports O1 and O2 were 9.57 μL min− 1. The flow rate toward the parallel channels (other outlets) is much smaller due to the hydrodynamic resistance caused by the much longer channel and bottlenecks. The simulation was calculated for empty channels because the back-pressure of the chromatographic packings is difficult to estimate by flow simulation. The backpressure of the chromatographic packings largely decreases the flow rate toward the parallel packings (and thus the volume of the sample plug). In Fig. 2.d and 2.e the pressure distribution and velocity magnitude can be examined in the complete channel system of the microchip. From these diagrams the relatively large differences in the flow velocities and the pressure between the different channels can be concluded. The differences between the flow rates can be illustrated also by plotting a line section (marked as dotted line in Fig. 2.a) perpendicular to the parallel channels (Fig. 2.f). As it was expected from the experimental observations (Video-1) the flow rates increased from one side to the other side, but the ratio of the maximal and minimal velocities (vmax/vmin) simulated in the parallel channels was amount to 3.4, slightly higher than in the experiments, which was measured to be 2.5. To understand what set of parameters could lead to this difference, the conditions of the transportation of the liquid in the chip were changed for the COMSOL simulations. The ports of the PDMS chips are created by punching the soft material. The dimension and the surface of the punched vertical channel and the obtained orifice at the end of the channel are rather occasional in microscopic scale. Smaller or larger bottlenecks can often form in the ports, which result in a small back-pressure against the liquid flow driven from port I. In a series of simulation experiments the values of the back-pressure at the port O4 were adjusted to 0 Pa, 0.68 Pa, 1 Pa, 6.9 Pa and 10 Pa. Increasing the back-pressure at port O4 the flow velocity difference of the parallel channels decreased (Fig. 3). However, after an application of back-pressure of around 2 Pa, instead of a monotonous increase of the flow rates from the left channels to right ones, the flow rates were the lowest in the middle channels and highest in the sidechannels (Fig. 3). Using 6.9 Pa back-pressure the flow rates in the two
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Fig. 2. COMSOL simulations of pressure distribution (b) and flow rates (c) in the sample introduction part (junction of fluid channels) and in the complete channel system (d, and e,) of the microchip. The flow rate applied at the inlet port I was 10 μL min−1, and the pressure at the outlet ports O1–O4 were set to 0 Pa. The line graph of the velocity profiles plotted in the points of a straight line (marked as dotted line in (a)) crossing each parallel channel perpendicularly (f).
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Channels Fig. 3. Maximum velocities along the centerline in the parallel channels obtained by COMSOL simulations using different back-pressures (0, 0.68, 1, 6.9, 10 Pa) at the outlet port O4. The flow rate applied at the inlet port I was 10 μL min−1, and the pressure at the outlet ports O1–O3 were set to 0 Pa. In case of the application of 6.9 Pa back-pressure at outlet port O4 the line graph is shown. The maximum velocity magnitudes from each parallel channel are calculated from the velocity profiles measured on the centerline (marked as dotted line in (Fig. 2.a)).
outermost (left and right) channels were equal, however the ratio of the highest and lowest rate was 1.2, which was found to be the best condition to reach similar flow rates. Increasing the back-pressure further, the flow rates become higher in the left-side channels (which are located closer to port O4). We suppose that the back-pressure in a range of 0– 1 Pa might be formed by the “non-perfect” dimension/shape of the outlet port. Although this pressure is less than 1% of the pumping pressure in the inlet port I, it has considerable effect on the distribution of the flow rate in the parallel channels. When the applied pressure at port O4 was changed from 0 Pa to 1 Pa with 0.01 Pa steps, the ratio between the maximum and minimum velocities was calculated. In this study it was found that the application of 0.68 Pa resulted in the same distribution of flow rates (when the ratio of the maximal and minimal flow rates was 2.5) that was experimentally obtained. The closing the port O4 did not help equalize the flow rates in the parallel channels, the vmax/vmin was found to be 1.9. If the O4 port is set as inlet (I2) with 10 μL min−1 flow rate similar to the original inlet (I1), the flow velocities gradually reduced from the left side to the right side and the vmax/vmin was found to 1.9. From these results it can be concluded that the presence of a small (just a few Pa) back-pressure can have a quite large effect on the velocity distribution. A small back-pressure can be generated by the difference of liquid heights (hydrostatic pressure) or bottlenecks in the ports. Because of the reproducible generation of a small (0–10 Pa), constant pressure in practical microfluidics is difficult, the adjustment of the back-pressure is not a proper way of equalizing the flow rates in the parallel channels. Pumping the liquid into the microchip through port I with different flow rates as 0.1 μL min−1, 1 μL min−1, 10 μL min−1 and 100 μL min−1 has practically no effect on the distribution of flow rates, the vmax/vmin values were 3.37, 3.37, 3.40, and 3.43, respectively. Although the Reynolds number increases proportionally with the increasing flow rate, the largest Reynolds number is only 0.2 (for the 100 μL min−1 pumping rate) showing the strong laminarity of the system. The flow profiles in the channels flatten with the reduction of the flow rate. Additionally, the equalization of the flow rates in the parallel channels was tried to achieve by the modification of the channel geometry. The shape of the straight-line channel before the bottlenecks was
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converted to a trapezoid shape one, where the ratio of the opposite parallel sides d1/d2 was set to 1:1, 2:1, 4:1, 8:1 (Fig. 4.a). Due to this trapezoid shape of the channel, the flow in front of the bottlenecks slowed down close to the longer side of the trapezoid (d1). When the ratio of the d1/d2 was changed to 2:1, the vmax/vmin value was improved (decreased) to 1.65, but if the d1/d2 was set to 8, the flow rates were equalized (vmax/vmin = 1.0) (Fig. 4.b). The velocity and pressure field diagrams for the microchip, where the ratio of the d1/d2 was 8, are shown in Fig. 4.c and 4.d. From the diagram of Fig. 4.c it can be concluded that at the acute angle of the trapezoid channel the flow rate of the liquid is almost zero, that is, the dead-volume of the trapezoid channel can be partly reduced by making it round as it was shown in Fig. 4.e. The previous simulations were made for the microchips that include exclusively open channels. When the parallel channels are homogeneously packed with chromatographic packing materials, the flow rates will be much smaller due to the high back-pressure, however the velocity distribution remains the same as in open channels. The microchip having multiple channels with equalized flow rates as the design optimized by COMSOL simulation was created with microfabrication and the parallel channels were uniformly packed with C18 modified silica particles of 5 mm (Fig. 5.a). Repeating the chromatographic separations of the two dyes in this microchip as it was shown in Fig. 1 and Video-1, now the flow rates in the 12 channels became very similar, the vmax/vmin equals to 1.10 (Fig. 5.d) (Video-2). The distribution of the flow rates can be simply determined based on the movement of both the firstly eluted yellow dye (Fig. 5.b) and the later eluted blue dye profiles (Fig. 5.c). In the microchips with 12 parallel chromatographic packings shown in Figs. 1–5 only one sample could be injected, which was then split
a,
to 12 equals parts before the bottlenecks. Since a large number of parallel and independent analytical systems can be arranged on a single microchip, the implementation of multiple chromatographic separations for several samples at the same time is probably also possible. Currently, a three-layer microchip design, which makes it possible to separately transfer samples from each sample reservoirs is being studied with the use of simulations. 4. Conclusions In microfluidic devices, especially if those include complex channel system, alterations between the experimentally gained and the expected (desired) flow rates at given parts in the microchip are often obtained. Without the use of simulation software it is quite difficult to get the channel pattern with the right dimensions or the best experimental parameters (flow rates, pumping pressure). Using simulation the exhausting preparation of microfluidic chip (including lithographic masks, molds) with trial and error method can be avoided. It was shown that COMSOL can help to better understand why the velocity distribution obtained in a microchip sometimes differs from that we expected. Our results also prove that a channel design optimized by COMSOL produces the same performance and characteristics as the experimental microfluidic chip fabricated in a laboratory. We designed a chip, in the parallel channels of which uniform velocities of the flow could be achieved, which is essential to provide for chromatographic separations. We studied the effect of opening or closing certain ends of the channels, application of pressure/back-pressure at several parts of the chip, but the best result was achieved with the modification of dimensions (shape) of the channel pattern. The COMSOL simulations matched well with the obtained experimental results.
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O4
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d,
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Fig. 4. Studies for equalization of the flow rates in the parallel channels by the modification of the channel pattern. The schematic drawing of the microchip, where the ratios of the d1/d2 (widths of the channel before the bottlenecks) were changed between 1 and 8 (a). The results of the simulation: velocity distribution in the channels using channel patterns with different ratios of the d1/d2 (b), velocity (c) and pressure distribution (d) diagrams for microchip where the ratio of the d1/d2 was 8.
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Fig. 5. Multiple chromatographic separations on microchip. The schematic representation (a) of the microchip with 12 parallel C18 packings which is identical to the one described in Fig. 1 except the channel before the bottlenecks has a trapezoid shape, where the ratio of the opposite parallel sides d1/d2 were set to 8:1. While the blue dye was completely retained at the beginning of the packing, the yellow dye was washed out by mobile water phase (b). Changing the mobile phase to methanol the blue dye was eluted (c). The linear flow rates in the parallel channels of the microchip were measured based on the video of the chromatographic elutions of the blue dye through the parallel packings (d).
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.microc.2015.05.019. Acknowledgments The authors gratefully acknowledge the financial support for this research by grants from the National Scientific Research Fund, Hungary (OTKA K75286), the EU and co-financed by the European Social Fund under the project SROP-4.2.2.B-15/1/KONV-2015-0001, the Pázmány University grant KAP-1.1-14/024 (recipient: Kristóf Iván) and the Hungarian NAP grant no.: KTIA-NAP_13-1-2013-0001. References [1] P. Tabeling (Ed.), Introduction to Microfluidics, University Press, Oxford, 2005. [2] J.P. Kutter, Liquid phase chromatography on microchips, J. Chromatogr. A 1221 (2012) 72–82. [3] J. Huft, C.A. Haynes, C.L. Hansen, Fabrication of high-quality microfluidic solid-phase chromatography columns, Anal. Chem. 85 (2013) 1797–1802. [4] J. Huft, C.A. Haynes, C.L. Hansen, Microfluidic integration of parallel solid-phase liquid chromatography, Anal. Chem. 85 (2013) 2999–3005. [5] A. Nagy, A. Gaspar, Packed multi-channels for parallel chromatographic separations in microchips, J. Chromatogr. A 1304 (2013) 251–256. [6] S. Cohin (Ed.), Microfluidics, Wiley, Hoboken, USA, 2010.
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