Microchip Injection and Separation Anomalies Due to Pressure Effects

While performing routine electroosmotically driven CE separations on microfluidic chips, we have observed peak shape, migration time, and baseline dri...
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Anal. Chem. 2001, 73, 4079-4086

Microchip Injection and Separation Anomalies Due to Pressure Effects H. John Crabtree,*,† Eric C. S. Cheong,†,‡ Daryle A. Tilroe,† and Christopher J. Backhouse‡

Micralyne Inc., 1911-94th Street, Edmonton, Alberta, T6N 1E6, Canada, and Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, T6G 2G7, Canada

While performing routine electroosmotically driven CE separations on microfluidic chips, we have observed peak shape, migration time, and baseline drift anomalies. Pressure-driven backflow (opposing electroosmotic flow (EOF)) has been observed and characterized, and meniscus surface tension (Laplace pressure) is cited as the likely cause. However, there are a number of interdependent factors that affect bulk flow in a microchip environment, including evaporation, buffer depletion due to hydrolysis, EOF pumping, siphoning, viscosity changes due to Joule heating, and Laplace pressure. Given the complexity of such a system, pressure effects were isolated from EOF, and to some extent, siphoning effects were isolated from suspected meniscus effects. Pressure flow observed in the absence of an applied field ranged from 0.4 to 0.8 mm/s, which was on the order of the EOF generated experimentally, 0.6 mm/s at a field of 150 V/cm, and was some 10-20 times larger than what would be predicted merely from a difference in liquid levels (siphoning). Furthermore, experiments were performed without an electric field and with the chip tilted so that meniscus flow ran “uphill” against a siphoning backflow and showed siphoning flow to have a negligible effect upon meniscus flow under the microchip conditions studied. These findings are relevant to the profusion of microfluidic and array-based technology that also use microliter liquid volumes in like-sized reservoirs with similar menisci. Since 1990 when Manz first described the concept and potential of miniaturized total analysis systems,1,2 or µTAS, microfluidic chips have been fabricated for and applied to electrophoretic,3-6 chromatographic,7-9 and hybrid separation techniques,10-15 as well * To whom correspondence should be addressed: (e-mail) [email protected]; (phone) (780) 431-4400. (fax) (780) 431-4422. † Micralyne Inc. ‡ University of Alberta. (1) Manz, A.; Fettinger, J. C.; Verpoorte, E.; Lu¨di, H.; Widmer, H. M.; Harrison, D. J. Trends Anal. Chem. 1991, 10, 144-149. (2) Manz, A.; Graber, N.; Widmer, H. M. Sens. Actuators 1990, B1, 244-248. (3) Manz, A.; Harrison, D. J.; Verpoorte, E.; Fettinger, J. C.; Paulus, A.; Lu ¨ di, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253-258. (4) Harrison, D. J.; Flurri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (5) Jacobson, S. C.; Hergenro ¨der, R.; Alvin W. Moore, J.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127-4132. (6) Jacobson, S. C.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1995, 67, 2059. 10.1021/ac010217r CCC: $20.00 Published on Web 08/04/2001

© 2001 American Chemical Society

as to non-separations-based microfluidic manipulations.16-21 The earliest example by Terry7 in the 1970s predates the past decade’s surge in µTAS research entirely. Microfabrication and miniaturization offer many inherent advantages which have been described at length in several reviews.22-24 Advantages include fast thermal diffusion,25 reduced band broadening, high levels of sequential process integration,26-28 integration of complex microfeatures for separation or detection systems,10,29-33 and capacity for highthroughput parallel processing.34 The challenges that arise in the microdomain include very sensitive detection of minute quantities of analyte being monitored, small volume, and often highly parallel (7) Terry, S. C.; Herman, J. H.; Angell, J. B. IEEE Trans. Electron Devices 1979, 26, 1880-1886. (8) Ocvirk, G.; Verpoorte, E.; Manz, A.; Grasserbauer, M.; Widmer, H. M. Anal. Methods Instrum. 1995, 2, 74. (9) Ericson, C.; Holm, J.; Ericson, T.; Hjerte´n, S. Anal. Chem. 2000, 72, 8187. (10) He, B.; Tait, N.; Regnier, F. Anal. Chem. 1998, 70, 3790-3797. (11) Kutter, J. P.; Jacobsen, S. C.; Matsubara, N.; Ramsey, J. M. Anal. Chem. 1998, 70, 3291-3297. (12) Oleschuk, R. D.; Shultz-Lockyear, L. L.; Ning, Y.; Harrison, D. J. Anal. Chem. 2000, 72, 585-590. (13) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468. (14) Moore, A. W., Jr.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1995, 67, 4184-4189. (15) Mao, Q.; Pawliszyn, J. Analyst 1999, 124, 637-641. (16) Fiedler, S.; Shirley, S. G.; Schnelle, T.; Fuhr, G. Anal. Chem. 1998, 70, 1909-1915. (17) Bessoth, F. G.; Mello, A. J. d.; Manz, A. Anal. Commun. 1999, 36, 213-215. (18) Xu, C.; Wang, Y.; Cao, M.; Lu, Z. Electrophoresis 1999, 20, 1829-1831. (19) Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.; McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426-430. (20) Figeys, D.; Ning, Y.; Aebersold, R. Anal. Chem. 1997, 69, 3153-3160. (21) Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 1997, 69, 1174-1178. (22) Becker, H.; Manz, A. Science Prog. 1996, 79, 49-63. (23) Kopp, M. U.; Crabtree, H. J.; Manz, A. Curr. Opin. Chem. Biol. 1997, 1, 410-419. (24) Manz, A.; Harrison, D. J.; Verpoorte, E.; Widmer, H. M. Adv. Chromatogr. 1993, 33, 1. (25) Kopp, M. U.; Mello, A. J. d.; Manz, A. Science 1998, 280, 1046-1048. (26) Waters, L. C.; Jacobsen, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 5172-5176. (27) Waters, L. C.; Jacobsen, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 158-162. (28) Woolley, A. T.; Hadley, D.; Landre, P.; Mello, A. J. d.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (29) Woolley, A. T.; Lao, K.; Glazer, A. N.; Mathies, R. A. Anal. Chem. 1998, 70, 684-688. (30) Drott, J.; Lindstro ¨m, K.; Rosengren, L.; Laurell, T. J. Micromech. Microeng. 1997, 7, 14-23. (31) Crabtree, H. J.; Kopp, M. U.; Manz, A. Anal. Chem. 1999, 71, 2130-2138. (32) Kenis, P. J. A.; Ismagilov, R. F.; Whitesides, G. M. Science 1999, 285, 8385. (33) Liang, Z.; Chiem, N.; Ocvirk, G.; Tang, T.; Fluri, K.; Harrison, D. J. Anal. Chem. 1996, 68, 1040-1046.

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sample introduction, as well as fluid flow dynamics35-37 owing to the multiple microchannel networks typical of microfluidic chips. Indeed, the increasing demand in academic and commercial arenas both for microfluidic devices to become more highly integrated and for microfluidic methodologies performed therein to become more reproducible will necessitate a more complete and fundamental understanding of fluid flow on chips. One particular fluid flow problem, which has been known to exist in both capillary- and chip-based capillary electrophoresis (CE) separations, is the coexistence of unwanted pressure-driven flow with electroosmotic flow (EOF). The presence of parabolic, pressure-driven flow detracts from the even radial profile of axial velocity generated by EOF. For capillary CE work, one merely ensures that the liquid levels in the ∼1-mL reservoirs into which the capillary ends are inserted are very nearly level to prevent a siphoning effect. Chip-based separations become somewhat more complicated in this regard, given a more complex network of connected channels and much smaller reservoir dimensions which are more susceptible to the effects of evaporation, buffer depletion, and Laplace pressures arising from surface tension. Laplace pressures38-40 are generated at meniscus interfaces and depend on the shape of the menisci. The pressure is larger on the concave side of the meniscus. In capillary rise experiments, this pressure difference causes water to rise up in the capillary when one end is inserted into a beaker of water until the hydrostatic pressure created by the (vertical) column of water equals the Laplace pressure difference. For a spherical meniscus surface, the equation for the Laplace pressure, ∆PL, is

∆PL ) 2γ/r

(1)

where γ is the surface tension at the liquid-air interface (force/ distance) and r is the radius of curvature of the meniscus, defined40 by

r ) rreservoir/cos θ

(2)

where rreservoir is the reservoir radius and θ is the contact angle. The effects of ionic strength and pH on EOF were studied previously with the capillary format41-44 and can be assumed to be similar if not identical in glass chips. Compensation for solvent evaporation in nanovial arrays was investigated on a chip format.45 (34) Shi, Y.; Simpson, P. C.; Scherer, J. R.; Wexler, D.; Skibola, C.; Smith, M. T.; Mathies, R. A. Anal. Chem. 1999, 71, 5354-5361. (35) Ermakov, S. V.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1998, 70, 44944504. (36) Seiler, K.; Fan, Z. H.; Fluri, K.; Harrison, D. J. Anal. Chem. 1994, 66, 34853491. (37) Paegel, B. M.; Hutt, L. D.; Simpson, P. C.; Mathies, R. A. Anal. Chem. 2000, 72, 3030-3037. (38) Adamson, A. W.; Gast, A. P. Physical Chemistry of Surfaces, 6th ed.; John Wiley and Sons: Toronto, 1997. (39) Hiemenz, P. C.; Rajagopalan, R. Principles of Colloid and Surface Chemistry, 3rd ed.; Marcel Dekker: New York, 1997. (40) Fletcher, P. D. I.; Haswell, S. J.; Paunov, V. N. Analyst 1999, 124, 12731282. (41) Belder, D.; Elke, K.; Husmann, H. J, Chromatogr., A 2000, 868, 63-71. (42) Salomon, K.; Burgi, D. S.; Helmer, J. C. J. Chromatogr. 1991, 559, 69-80. (43) Lukacs, K. D.; Jorgenson, J. W. J. High-Resolut. Chromatogr. Chromatogr. Commun. 1985, 8, 407-411. (44) Issaq, H. J.; Janini, G. M.; Chan, K. C.; Rassi, Z. E. Adv. Chromatogr. 1995, 35, 101-169.

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Siphoning arising from different liquid levels in reservoirs was examined experimentally on a microchip by Boer and coworkers.46 Meniscus/Laplace pressure phenomena are used to advantage for passive valving in the growing number of rotating CD-format microfluidic devices being developed by companies such as Gyros Microlab47 and Gamera Bioscience.48 In these devices, centripetal acceleration is used to generate fluid flow, and channel dimensions are designed to halt fluid flow up to threshold pressures generated by the rotating disk. In-channel meniscus radii are perhaps 1-2 orders of magnitude smaller than in our reservoirs, so the Laplace pressures are much higher. Fletcher and co-workers40 indicated theoretical considerations regarding Laplace pressures at reservoir menisci for open-channel CE separations, but to our knowledge, there is no experimental evidence characterizing these phenomena arising from millimetersized reservoirs and menisci. We present experimental conditions for chip-based CE work under which reservoir Laplace pressures (and other effects) will tend to be present and degrade a separation, as well as experimental evidence suggesting that these effects are largely due to Laplace pressures. EXPERIMENTAL SECTION Apparatus. A block diagram of the Micralyne Microfluidic Tool Kit instrument (Micralyne Inc., Edmonton, AB, Canada) is shown in Figure 1; in essence, the kit consists of high-voltage (HV) power supplies coupled with a laser-induced fluorescence (LIF) detection system. As configured for these experiments, the HV electronics comprised two HV boards, each of which has two +6-kV power supplies capable of 80 µA, sink or source. The outputs from the HV boards are switched by reed relays between ground, floating, and HV states and are connected to Pt electrodes in a grid above the chip stage. The green LIF detection module was chosen for these experiments, in accordance with the fluorophore used (TAMRA). The epiluminescent confocal detection module makes use of a 40×, 0.55 NA lens to focus the beam from a frequency-doubled Nd:YAG diode-pumped solid-state laser (532 nm) onto a channel on the chip to excite any suitable fluorophores. Fluorescence is collected with the same objective, deflected with a dichroic mirror toward either the eyepiece or the PMT, depending on which is rotated into the optical axis. The PMT optical path has a 10-nm band-pass filter centered at 568.2 nm. Signal from the PMT is then collected on the optics board with 16-bit resolution at 200 Hz. For the purpose of measuring currents, digital multimeters were connected serially along each electrode lead (NB: avoid contact with multimeters in this configuration, as they float with the high voltage delivered at the electrode); currents could be measured to 0.1 µA. (45) Litborn, E.; Emmer, Å.; Roeraade, J. Electrophoresis 2000, 21, 91-99. (46) Boer, G.; Dodge, A.; Flurri, K.; van der Schoot, B. H.; Verpoorte, E.; de Rooij, N. F. Studies of Hydrostatic Pressure Effects in Electrokinetically Driven µTAS, Micro Total Analysis Systems ′98, Banff, Alberta, Canada 1998; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1998; p 492. (47) Ekstrand, G.; Holmquist, C.; O ¨ rlefors, A. E.; Hellman, B.; Anders, L.; Andersson, P. Microfluidics in a Rotating CD, Micro Total Analysis Systems 2000, Enschede, Holland 2000; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2000; p 623. (48) Duffy, D. C.; Gillis, H. L.; Lin, J.; Sheppard, N. F., Jr.; Kellogg, G. J. Anal. Chem. 1999, 71, 4669-4678.

Figure 1. Schematics of the Micralyne SC microfluidic chip and the Micralyne Microfluidic Tool Kit, or µTK. (A) Microfluidic chip layout consists of two channels, 8 and 85 mm long, each 20 µm deep and 50 µm wide, intersecting 5 mm along the long channel, midway across the short channel. Each channel is terminated by 1.1-mm-deep, 2-mm-diameter reservoirs. (B) The µTK consists of software-controlled high-voltage power supplies (4) and laser-induced fluorescence detection designed for microfluidic chips.

The instrument is connected to a PC through the serial port, and the HV, laser, and PMT functions are controlled with a LabView (National Instruments, Austin, TX) executable file. Micralyne SC microfluidic chips, a schematic of which is also shown in Figure 1, were used to perform all electrophoretic and pressure flow experiments. The 16 × 95 × 2.2 mm glass chips consist of a cross-shaped intersection of a short and a long channel, 8 and 85 mm long, respectively; the intersection occurs 5 mm from one end of the long channel, bisecting the short channel. Channels are D-shaped in cross section: 20 µm deep, 50 µm wide at the top. Each channel has a 2-mm-diameter, 1.1mm-deep well at each end; the wells are labeled sample reservoir (SR), sample waste (SW), buffer reservoir (BR), and buffer waste (BW) as depicted in Figure 1. Reagents and Solutions. Tris-borate-EDTA (TBE) buffer solution was purchased at the 5× concentration (445 mM in both tris(methoxy)aminomethane and boric acid, 10 mM in ethylenediaminetetraacetic acid, Sigma-Aldrich), diluted volumetrically to 1× TBE and other, more dilute concentrations with deionized water, and finally filtered through 0.45- or 0.2-µm filters. TAMRA (Molecular Probes, Eugene, OR) was dissolved in MeOH at a concentration of 3.67 mM as a stock solution. This was then diluted to a 36.7 µM solution in 1× TBE and 10% (by volume) MeOH and was used as the dye sample solution for all subsequent runs involving TAMRA.

Cleaning solutions of 1.55 M HNO3 and 1.01 M NaOH were prepared from concentrated nitric acid (69% HNO3, Anachemia) and solid NaOH pellets (Fisher), respectively, and deionized water. Preparatory and Experimental Procedure. (1) Chip Preparation/Storage. To prepare the chip, cleaning fluids were drawn into the chip by applying vacuum to one reservoir and supplying the other three with the appropriate fluid. For a given fluid rinse, vacuum supplied by a 60-mL syringe was applied first to the BR reservoir for ∼1 min to visually observe rapid depletion of the volumes in adjacent wells and second to BW for 10 min to slowly flush the chip. Daily chip preparation consisted of drawing through first 1.55 M HNO3, then 1.01 M NaOH, and finally the running buffer, 1× TBE. When not in use, the chip was immersed in water with vacuum applied to BW to provide water rinsing. (2) CE Experiments. For electrophoretic experiments, sample solution was then loaded into SR and 1× TBE to the other three reservoirs, and the chip was ready to run; solutions were loaded into the reservoirs with 10-µL syringes (Hamilton Bonaduz, Switzerland). The volumes used were 3 µL in each well for the preliminary experiments. A series of 10 on-chip injections were performed by repeating the 80-s voltage program in Table 1 for each injection (800 s total). The first program step forms a sample plug in the intersection with EOF “pinching”, and the second injects the plug down the separation channel with EOF “pull-back” toward the SR and SW reservoirs. Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

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Table 1. Two-Step High-Voltage Program Used To First Form a Plug of Sample at the Channel Intersection and Then Inject It Down the Separation Channel toward the Detectora reservoir potential (kV) step

sample

buffer

sample waste

buffer waste

duration (s)

plug formation separation

1.5 5.4

1.1 6

GND 5.4

2 GND

20 60

a These two steps were repeated consecutively for replicate injections, as described in the Experimental Section.

(3) CE “Top-Up” Experiments. In the top-up experiments, 3 µL of dye solution was applied to SR, 3 µL of 1× TBE to SW, and 1.5 µL of 1× TBE to BR and BW; after five on-chip injections as per Table 1, an additional 3 µL was added to BR, and a second series of five injections was performed. The top-up experiments were performed both with the PMT in position to obtain an electropherogram and subsequently with the eyepiece in position to observe visually. (4) EOF Measurements. For the EOF measurement experiments, the procedure of Huang et al. was followed.49 The channels and three of the four wells on the chip were filled with 1× TBE and then 0.5× or 0.75× TBE was added to either the BR or BW well (3 µL of solution in each well). Three kilovolts was applied to BR, BW was grounded, the short channel was left floating, and current readings were recorded for 3 min at ∼5-s intervals using a stopwatch. As the more dilute solution replaced the 1× TBE, the current dropped linearly with time, until the dilute buffer had completely filled the channel, after which point the current readings formed a plateau. The intersection of the two linear portions of the data indicates the time, t, required to fill the channel. Given the channel length, L, the EOF velocity, veo, is determined as veo ) L/t; given applied voltage, V, the electroosmotic mobility, µeo may be determined through the equation

veo ) µeo(V/L)

(3)

(5) Field-Free Experiments. For the field-free experiments, three sets of experiments were conducted: the first with the instrument level; the second with the instrument tilted at an angle of ∼7.8°, which is sufficient to position one reservoir along the short channel, SW, below the other, SR; and the third with the instrument tilted at an angle of ∼20.1°, positioning SW below both BR and BW along the long channel, as well as below SR. These two angles correspond to approximate height differences of 1.3 and 3.5 mm, respectively, for reservoirs filled to the same extent; the exact equivalent height differences, which account for different meniscus volumes and shapes, have not been calculated, since the meniscus shapes are not known (and are not hemispherical). Tilting the chip allowed any flow induced by meniscus effects to be directly opposed by gravity. After having flushed the chip with 1× TBE, three of the reservoirs were filled to the same volume (either 0.5, 1.0, 2.0, or 2.5 µL), leaving one reservoir (SW) at the (49) Huang, X.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1988, 60, 1837-1838.

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Figure 2. Example of a series of 10 successive TAMRA dye injection/separation 80-s sequences. (A) Raw data; the baseline can clearly be seen to drift after the seventh injection. (B) Parsed data; except for the first injection, the dye peak appears later and with more band broadening with successive injections. Note that the abscissa in (B) includes the 20-s plug formation step.

end of the short channel empty; the chip was then mounted in the instrument and the LIF detection spot moved to the center of the channel intersection. Three microliters of the TAMRA sample solution was then added in ∼0.5 s to SW, and the data acquisition was started immediately after the injection. This produced a surge in the PMT signal when the fluorophore reached the intersection, usually some 2-15 s later; PMT signal versus time was recorded to show these breakthrough curves. Three field-free control experiments were also run (for 0°, 7.8°, and 20.1° instrument angles) exactly as described above, but with different volumes of reagents: 1.5 µL of TAMRA was applied to SW and 3 µL of 1× TBE elsewhere. No fluorescence breakthrough curve was expected. RESULTS AND DISCUSSION The data depicting the separation anomalies that inspired this investigation are shown in Figure 2. While no true separation occurs with the single-component sample used here, the reader may consider the term to represent the stage in the protocol that would analyze a multicomponent sample, were one used. Ten repetitions of the injection/separation sequence of Table 1 were performed, and after the seventh injection, the baseline can clearly be seen to drift. The elution order of the peaks typically resembles what is shown, namely, that peaks come out more slowly as the injection sequence progresses. Note that the more slowly eluting peaks are significantly more band broadened. Assuming the detection system is functioning properly, the most likely source for a gradually rising baseline is leakage of dye-laden solution from either branch of the short channel during the separation stage. To verify this postulate, the experiment was repeated with the fluorescence at the intersection monitored visually. Indeed, after the sixth injection, some fleeting dye leakage was observed emanating from the SW channel, and for the seventh

Figure 3. Illustration of backflow effect observed visually. (A) For the first injections, a nicely formed plug at the intersection injects cleanly into the long channel. (B) For injections made later on in the sequence, the plug forms nicely, but leakage is seen from the sample waste reservoir, despite the current and hence EOF opposing it in that channel (red arrows show dye flow opposing EOF in boxes highlighted in green).

and successive injections, that leakage became sustained and more pronounced; this is illustrated in Figure 3. Also shown are the directions of current flow and the approximate heights of fluids in the reservoirs, discussed next. The current values at the onset of each step in the voltage program were recorded and clearly showed that electroosmotic flow continued in the intended direction in each channel for both plug formation and separation steps, throughout the sequence of injections (data not shown). Thus, the backflow of dye solution in the SW channel of Figure 3 observed during the later separation stages occurred despite opposing EOF used for pull back. The reservoir height discrepancies at the end of a series of injections shown in Figure 3 suggest pressure effects as a possible cause for the backflow observed. Using the bottom of the meniscus as a volumetric indicator (an approximation, since the meniscus does not translate up and down, as in a pipet, but rather changes shape, in response to changing liquid volumes), the SW reservoir was about half full, the BW was about one-third full, while SR and BR were very nearly dry after 10 injections. The creation of these height discrepancies is consistent with EOF flow patterns. In essence, applying the program of Table 1 resulted in liquid being electroosmotically pumped from SR to SW during the injection stage and from BR to BW during the separation stages; with these pumping directions in mind, the height discrepancies noted are not surprising. Further, the existence of these height discrepancies is consistent with pressure-driven backflow patterns. Specifically, pressure-driven flow (be it from Laplace pressure or siphoning) from SW and BW is consistent with the anomalies observed late in the 10-injection sequence of Figure 2 when the discrepancies become pronounced, namely: (1) backflow from SW causes dye solution leakage and thus baseline drift, and (2) backflow from BW cause the TAMRA peaks to migrate more slowly and become more band broadened. The anomalies were suspected to be due to pressure effects, so a two-stage top-up experiment was performed wherein the anomalies were both induced and corrected by control of the liquid

volumes in reservoirs. In the first five-injection “deterioration” stage, the anomalies were induced by applying buffer and dye solutions to the chip as for Figure 2 except that only 1.5 µL of buffer (instead of 3 µL) was applied to BR and BW to bring about dye backflow into the long channel. In the second “correction” stage, the run was stopped, a 3-µL top-up was applied to BR to reverse the effects caused in the first stage, and five more injections were performed. The results are shown in Figure 4. In Figure 4A and B, the onset of baseline drift can clearly be seen by the fifth injection in the “deterioration” stage (not seen until the seventh injection with 3-µL volumes in all reservoirs), along with significant peak delay. Panels A and C of Figure 4 show that, after the top-up to BR, the baseline drift is eliminated in the next injection (6), and the TAMRA zones migrate more rapidly in subsequent injections. This is consistent with the elimination of pressure backflow from SW and BW. While the evidence presented in Figures 2-4 above suggests pressure-induced backflow, it is by no means conclusive; a number of different and interrelated factors are brought to bear on pressure-driven and electroosmotically driven fluid flow in the channels during a series of microfluidic manipulations and should be considered. First, as mentioned before, evaporation occurs at all reservoirs simultaneously and, by decreasing the volume, will concentrate all solutions. Over the span of a 10-injection sequence, about twothirds of the total reservoir liquid volume is lost to evaporation. Such depletion will increase the ionic strength and, hence, decrease EOF (Salomon et. al have written on the dependence of EOF on buffer strength42). By changing the solution concentration and the liquid volume in the reservoir, this factor will directly affect Joule heating, siphoning, and Laplace pressures, described below. Second, buffer depletion is occurring at all reservoirs that are passing a current. Specifically, hydrolysis of water produces H+(aq) and O2(g) at anodes (positive current) and HO-(aq) and H2(g) at cathodes (negative current). The amount of net current passed over the span of a 10-injection sequence can be significant, Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

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linear flow rate due to height differences in a D-shaped microchannel, vp, can be quantified40,46 with the equation

vp ) ∆hFgDh2/(2 × 15.626)ηL

Figure 4. Migration time and baseline drift anomalies induced and corrected by reservoir volumes in a two-stage experiment. In (A), fiveinjection “deterioration” stage begun as in Figure 2 except for reduced volumes in BR and BW, which hastens the onset of pressure backflow and hence baseline drift and peak delay anomalies. After stage 1, a 3-µL top-up is applied to BR, and the second five-injection “correction” stage is begun, reducing baseline drift and peak delay. Data in frames (B) and (C) are as in frame (A), but are parsed and magnified to highlight the onset and correction of baseline drift and peak delay. Note that the abscissa axis in (B) and (C) includes the 20-s plug formation step.

compared to the buffering strength of the solution used (3 µL of 1× TBE). On the basis of the currents recorded for all 20 steps of a sequence, and ignoring all other effects, 38.8 mM H+ is produced at BR, the reservoir with the greatest net current (data not shown). As buffer depletion is approached, the pH may be expected to change dramatically in the direction in which the buffer was depleted and, thereby, significantly change the EOF.42,43 In some of the 10-injection sequences performed, the migration time increased by as much as 50% (12-18 s); if we attribute this change in net velocity only to diminishing EOF, then EOF along the long channel would have decreased from 3.1 to 2.5 mm/s, a decrease of only 19% (note that the dye’s net mobility is the sum of its opposing electrophoretic and electroosmotic mobilities). By changing the EOF pumping speed, this factor will directly affect electroosmotic pumping volumes, described next. Third, the current being passed is to generate electroosmotic pumping, which will affect the volume of all reservoirs. Again, ignoring all other effects, the volumetric depletion over 10 injections seen at BR due to EOF is 2.6 µL, based on an experimentally determined49 value of µeo of 4.4 × 10-4 cm2/(V‚s) (data not shown). By changing the liquid volume in the reservoir, this factor will directly affect buffer depletion, described above, as well as siphoning and Laplace pressures, both described below. Fourth, differences in liquid level heights between reservoirs will cause siphoning from reservoirs with higher liquid levels. The 4084

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(4)

where ∆h is the difference in liquid levels, F is the density of the solution, g is the acceleration due to gravity, Dh is the hydraulic diameter (Dh ) 4 (cross-sectional channel area/cross-sectional channel perimeter)), η is the solution viscosity, and L is the channel length. As a measure of this effect, one could consider the limiting case of flow being generated by a full 1.1-mm-high SW reservoir on our chip, with all other reservoirs empty. The flow thus passes through a 4-mm channel and then splits at the intersection to three channels 4, 5, and 80 mm long; the three parallel paths produce a parallel flow resistance equivalent to a 2.2-mm channel, so the total channel length, L, can be taken as 6.2 mm. For our channels and experiments, Dh ) 27.0 µm, F ) 1.0 g/mL, and the viscosity, η, is taken to be that of water at 20 °C, 1.0 cP. Given these values, a flow of 41 µm/s would result. In contrast, the EOF flowing in the opposite direction during each separation step of a sequence is much larger, at least initially; given a field at that stage in that channel of ∼150 V/cm, an EOF of ∼660 µm/s is produced. In terms of height, an 18-mm column would be required to counter the EOF. While liquid level differences and hence the flow they generate are initially small, and are dwarfed by full-strength EOF, they may become significant later in a sequence when larger and when opposing EOF has weakened. By changing the liquid volume in the reservoir, this factor will also directly affect buffer depletion, described above, and Laplace pressure, described below. Fifth, Joule heating arising from the applied electric field will induce both a radial temperature gradient in the channel and an overall increase in the temperature of the fluid and, hence, a decrease in its viscosity. Since both the electroosmotic and electrophoretic mobilities vary inversely with viscosity, an increase in migration speed and, hence, a decrease in migration time would be observed. No such trend was observed, and usually migration times increased, indicating that this was not a factor. By changing the EOF pumping speed, this factor will directly affect electroosmotic pumping volumes, described above. Sixth, Laplace pressures, surface tension, or surface free energy plays a role in pressure-driven flow. Laplace pressures, as described by eqs 1 and 2 in the introduction, are generated at meniscus interfaces. The magnitude of pressure generated at one of our wells can be estimated using eqs 1 and 2, using the surface tension of water (72.8 mN/m), a reservoir meniscus of 1 mm, and a contact angle of 0° against glass; a value of 146 Pa is obtained, which corresponds to a height difference of ∼15 mm. This pressure differential would be observed if all the other reservoirs were exactly full and had flat menisci; often, similar menisci are present in all reservoirs, and so opposing Laplace pressures cancel each other out. The calculation of an exact Laplace pressure at a reservoir on our chip (and likely on many other microfluidic devices) is encumbered by a number of considerations: (1) the meniscus profile is not always spherical, so that eqs 1 and 2 are not accurate; (2) the reservoir depth is the same as its radius, so that as the volume in the reservoir changes, the meniscus profile changes

Figure 6. Pressure-driven flow with no electric field. (A) Replicate breakthrough curves for dye traveling 4 mm from reservoir to LIF detection at intersection; 0.2 V dashed line represents threshold for all breakthrough curves at which front was deemed to have arrived. (B) Variation of dye front arrival time with volume of buffer in three other reservoirs, and with chip angle as per Figure 5. Dye front moves quickest with smallest opposing buffer volumes; opposing Laplace pressure flow with siphoning by raising the chip has little effect on frontal arrival times.

Figure 5. Meniscus vs siphoning with no electric field. Chip set level to align meniscus and siphoning flow in same direction (left to right) or tilted to oppose meniscus flow (left to right) with siphoning flow (right to left). Tilting the chip 7.8° brings the SW reservoir containing the dye solution completely beneath SR, while tilting it 20.1° brings SW beneath all other reservoirs, as shown by the dashed level line.

(a fixed meniscus profile does not translate up and down the reservoir, as in a capillary); (3) as discussed above, liquid volumes on the order of the reservoir volume are consumed by electroosmotic pumping and evaporation, so it should be assumed that meniscus shapes are changing dramatically during the course of the experiments described thus far; (4) as recognized by Fletcher et al.,40 both the insertion of electrodes in the liquid in the reservoirs and their relative positioning within will affect the meniscus shape. A qualitative thermodynamic argument may be made to describe the direction of flow based on the surface free energy of the menisci. As the liquid volume in the well decreases, the meniscus is observed to stay anchored at the top edge of the reservoir, and the curved surface of the meniscus is seen to approach the bottom and sides of the reservoir (Figure 3). In so doing, the surface area of the meniscus must increase by virtue of the work done on it by electroosmotic pumping. To minimize the surface free energy, the liquid surface area in all the reservoirs will be minimized, directing flow toward the more empty reservoirs until menisci are equal. The menisci may thus be regarded as “elastic” surfaces which pull liquid toward them until countered by equal forces elsewhere. By changing the liquid volume in the reservoir, Laplace pressure-driven flow will affect buffer depletion and siphoning, discussed above.

Given the above discussion, which highlights the multifactorial nature of chip-based fluid flow, and specifically the difficulty of attributing fluid flow to one factor among many acting simultaneously, a further series of electrical field-free experiments was conceived to isolate both pressure-driven flow from electroosmotic flow, as well as siphoning flow from meniscus-driven flow. The experiments measured the arrival time of dye solution at the channel intersection after being added to either SW or SR. The variables examined were different volumes of buffer in each of the three other reservoirs opposing the 3 µL of dye, as well as different angles to which the chip was tilted, to oppose Laplace pressure-driven flow with siphoning. Figure 5 shows the chip positioning in the tilted chip experiments, and Figure 6 shows the data for all the electrical field-free experiments. In Figure 6A, typical replicate breakthrough curves for dye traveling the 4 mm and arriving at the channel intersection are shown. Variability in replicate breakthrough curves likely arises from inconsistencies in delivering the 3-µL volume of dye with a 10-µL syringe and subsequent triggering of data acquisition, both done by hand. For this reason, the threshold at which the dye was considered to have arrived was chosen to be low (0.2 V), to mark the front arrival early on. Figure 6B shows these threshold arrival time data plotted against opposing buffer volume for different chip tilt angles. Even with a low threshold marking frontal arrival, the data scatter owing to the nonautomated procedures mentioned is considerable. Control experiments with 1.5 µL of dye opposed by 3.0 µL of buffer were also conducted with the chip level and at each angle; no fluorescence breakthrough curve was seen in the data collected (data not shown). The data of Figure 6B, while lacking in precision, do show two general trends. The first is that, for a level or tilted chip, the Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

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speed of the dye front decreases as the opposing buffer volume is increased from 0.5 to 2.5 µL. While it is intuitively tempting to attribute this flow rate trend to siphoning arguments, the discussion of eq 4 above showed that a maximum flow rate of 41 µm/s could be achieved by siphoning; this represents a minimum arrival time of 98 s. The slowest and fastest average arrival times of 9.5 and 4.9 s using a level chip were seen with 2.5 and 0.5 µL of buffer in the non-dye reservoirs, respectively, which is some 10-20 times faster than what is calculated for siphoning. The second trend that can be seen in comparing tilted chip to level chip data in Figure 6B is that the arrival times do not increase appreciably, if at all, when the chip is tilted. The angles chosen and illustrated with dashed lines in Figure 5 correspond to lowering the SW reservoir (containing the dye) beneath SR at 7.8°, and beneath BR/BW at 20.1°, which requires that any siphoning flow oppose the flow of dye from SW to the intersection, regardless of the volumes contained in any of the reservoirs. The observation that the dye flow is not significantly affected by opposing siphoning confirms that siphoning flow is relatively insignificant under these conditions versus Laplace pressure- or meniscus-driven flow. Considering Laplace pressure alone as measured by the level chip breakthrough curves, the fast 4.9-s arrival time corresponds to a dye flow velocity of 0.81 mm/s and, through eq 4, to an equivalent 22 mm of water head or 216 Pa. Likewise, the slow 9.5-s arrival time corresponds to a dye flow velocity of 0.42 mm/s and to an equivalent 11 mm of water head or 112 Pa. These values are in rough agreement with the 15 mm or 146 Pa values calculated earlier from eqs 1 and 2. They are also similar in magnitude (but opposite in direction) to the electroosmotic flow of 0.66 mm/s at 150 V/cm toward SW and SR produced during the separation step. This numerical agreement suggests that the dye leakage and peak shape anomalies highlighted in this study are well described by Laplace pressure-driven flow. To summarize the results above, we observe the following: (1) dye backflow into the separation channel from the higher volume SW reservoir is seen after several replicate CE dye injections (Figures 2 and 3); (2) this backflow can be remedied by topping up the buffer volume in the BR reservoir (Figure 4); (3) pressure flow of dye in an electric field-free environment to the channel intersection is 10-20 times faster than siphoning calculations would predict; (4) opposing this flow with siphoning did not stop it or alter it appreciably (Figure 6B); (5) the pressure flow of dye in the electric field-free experiments is on the order of what is calculated for Laplace pressures for a reservoir of our dimensions; (6) this same pressure flow is on the order (opposite direction) of the experimentally determined EOF used to inhibit dye backflow when such backflow was observed in the CE experiments. When taken together, these experimental and theoretical findings strongly suggest that the anomalies observed while CE manipulations are performed on a microfluidic chip are owing to Laplace pressure-driven flow. CONCLUSIONS While electroosmotically controlled fluid flow on a microchip is a significant advantage insofar as CE separations, multichannel fluid flow, and valving are concerned, a number of different factors

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may detract from the flat frontal profiles, low band broadening, and electric control that make it so attractive. Several of these factors have long been known to exist, such as siphoning, evaporation, buffer depletion, and Joule heating. However, the effects of surface tension at menisci on microchips are seldom dealt with in the literature. We have identified and characterized a number of anomalies relating to baseline drift, peak shape, and migration time that occur during the course of routine microchip-based CE separations. Such anomalies become pronounced when (1) several microfluidic operations are performed in series so that significant volumes of liquid evaporate or are pumped preferentially toward selected reservoirs and when (2) these volumes are on the order of those in the reservoirs. Laplace pressure-driven or meniscus surface tension-driven flow was both present and significant in magnitude, on the same order as electroosmotic flow. Observations of baseline drift, peak broadening, and migration delay could be described using Laplace pressure under the conditions investigated. Subsequent experiments would help to clarify the role of Laplace pressure-driven flow in microfluidic manipulations by probing modified solution surface tension (e.g., with surfactants), as well as meniscus shape changes in response to both changing solution volume, and an electrode insertion. Unwanted pressure-driven flow in an open CE chip system may be remedied simply, such as by limiting experimentation in time and/or current consumption, by topping up reservoirs with solvent to limit evaporation and buffer depletion, or by using larger reservoirs to dramatically reduce the buffer depletion and relative volumetric depletion of the reservoir solutions. Another idea that has been suggested is to remove or alter the liquid-gas interface. For example, the use of a second, immiscible liquid phase (such as an oil) above the aqueous phase in the reservoir acts to inhibit evaporation; alternatively, a closed-loop, pump-driven system, analogous to the valve-controlled capillary manifold system commercialized by Convergent Bioscience, could be applied to a microfluidic manifold to control pressure-driven flow. Naturally, the limitations and procedures just described do not address all of the fluid flow factors discussed above and may not be appropriate for a particular chip-based analytical protocol. For applications where lengthy parallel or serial analyses of smallvolume “open” samples are to be implemented on microfluidic devices, Laplace pressure-driven flow phenomena should be considered. ACKNOWLEDGMENT The authors would like to thank Mark Munroe, Herb Hartshorne, and Bob Lyons at Micralyne and John Bowman’s group in the Department of Mathematics at the University of Alberta for helpful discussions. This work was supported by Micralyne, the University of Alberta, and by a grant from the Alberta Cancer Board (C.J.B.).

Received for review February 20, 2001. Accepted June 18, 2001. AC010217R