Numerical Analysis and Experimental Estimation of a Low-Leakage

Anal. Chem. 2003, 75, 5790-5796

Numerical Analysis and Experimental Estimation of a Low-Leakage Injection Technique for Capillary Electrophoresis Lung-Ming Fu† and Che-Hsin Lin*,‡

Graduate Institute of Materials Engineering, National Pingtung University of Science and Technology, Pingtung, Taiwan, 912, and Department of Mechanical and Electro-Mechanical Engineering, National Sun Yat-sen University, Kaohsiung, Taiwan, 804

This paper presents an experimental and numerical investigation into the use of low-leakage injection techniques to deliver sample plugs within electrophoresis microchips. The study addresses the principal material transport mechanisms such as electrokinetic migration, fluid flow, and diffusion and gives detail analyses to the double-L injection technique, which employs electrokinetic manipulations to avoid sample leakage within the microchip. Electrical potential contour, velocity vector, and streamline distribution in the micro CE chip are successfully developed. Experimental and numerical testing results show the double-L injection technique is capable of reducing sample leakage within cross-form microfluidic chips. The current study confirms the double-L injection technique has an exciting potential for use in high-quality, high-throughput chemical analysis applications and in many other applications throughout the field of micro total analysis systems. Microfluidic devices have been used successfully in a variety of separation techniques involving chemical or biological components. A typical application of such devices is the separation of DNA in genetic engineering processes. Micromachining technology now permits the fabrication of a network of microchips on a single quartz, glass, or plastic (PDMS, PC, PMMA) substrate, which can then be used to perform a variety of procedures, including sample handling, pretreatment, chemical reaction, and separation.1-5 These devices are frequently used in the separation of samples using a technique similar to that used in conventional capillary electrophoresis analysis, but more efficient and faster.6-8 * Corresponding author. E-mail: [email protected]. Tel: +886932840730. Fax: +886-946526044. † National Pingtung University of Science and Technology. ‡ National Sun Yat-sen University. (1) Culbertson, C.; Ramsey, R.; Ramsey, J. M. Anal. Chem. 2000, 72, 22852291. (2) Auroux, P. A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637-2652. (3) Wang, J.; Chatrathi, M. P. Anal. Chem. 2003, 75, 525-529. (4) Ramsey, J. M. Nat. Biotechnol. 1999, 17, 1061-1062. (5) Varpoorte, E. Electrophoresis 2002, 23, 677-712. (6) Schrum, D. P.; Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1999, 71, 4173-4177. (7) Auroux, P. A.; Iossifids, D.; Reyes, D.; Manz, A. Anal. Chem. 2002, 74, 2637-2652.

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The performance improvement is largely attributed to the very small volume of the injected sample plugs. These samples can often be less than half the volume of the samples used in the conventional capillary electrophoresis technique. A large number of researchers have investigated the injection and separation of samples within microfluidic chips. Traditionally, these devices use electrokinetic pumping techniques to manipulate the samples and incorporate microfluidic channels and mixing schemes operated by miniature injection valves and pumps.9,10 Several different valve configurations have been developed for the delivery of discrete samples, including the T-form,11,12 the crossform,13-16 the double-T form,17-20 and the pinched-valve form.21-25 Accordingly, more complex injection system configurations have been developed, including the multi-T form26 and the double-cross form27,28 injection systems, which are capable of providing variablevolume sample plugs. Jacobsen et al. proposed a gated injection (8) Mistry, K.; Krull, I.; Grinberg, N. J. Sep. Sci. 2002, 25, 935-958. (9) Emneus, J.; Yakvleva, J.; Davidsson, R.; Lobanova, A.; Eremin, S.; Laurell, T.; Bengtsson, M. Micro Total Anal. Syst. 2001, 432-434. (10) Leach, A. M.; Wheeler, A. R.; Zare, R. N. Anal. Chem. 2003, 75, 967-972. (11) Harrison, D. J.; Manz, A.; Fan, Z.; Ludi, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (12) Bianchi, F.; Ferrigno, R.; Girault, H. H. Anal. Chem. 2000, 72, 1987-1993. (13) Jacobson, S. C.; Hergenroder, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127-4132. (14) Ermakov, S. V.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 2000, 72, 35123517. (15) Crabtree, H. J.; Cheong, E. C. S.; Tilroe, D. A.; Backhouse, C. J. Anal. Chem. 2001, 73, 4079-4086. (16) Jin, Y.; Luo, G. A. Electrophoresis 2003, 24, 1242-1252. (17) Koutny, L. B.; Schmalzing, D.; Taylor, T. A.; Fuchs, M. Anal. Chem. 1996, 68, 18-22. (18) Khandurina, J.; Chovan, T.; Guttman, A. Anal. Chem. 2002, 74, 1737-1740. (19) Backofen, U.; Matysik, F. M.; Lunte, C. E. Anal. Chem. 2002, 74, 40544059. (20) Ocvirk, G.; Munroe, M.; Tang, T.; Oleschuk, R.; Westra, K.; Harrison, D. J. Electrophoresis 2000, 21, 107-115. (21) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 3212-3217. (22) Ermakov, S. V.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1998, 70, 44944504. (23) Schrum, D. P.; Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1999, 71, 4173-4177. (24) Alarie, J. P.; Jacobson, S, C.; Culbertson, C. T.; Ramsey, J. M. Electrophoresis 2000, 21, 100-106. (25) Alarie, J. P.; Jacobson, S. C.; Ramsey, J. M. Electrophoresis 2001, 22, 312317. (26) Fu, L. M.; Yang, R. J.; Lee, G. B.; Liu, H. H. Anal. Chem. 2002, 74, 50845091. (27) Yang, R. J.; Fu, L. M.; Lee, G. B. J. Sep. Sci. 2002, 25, 996-1010. (28) Fu, L. M.; Yang, R. J.; Lee, G. B. Anal. Chem. 2003, 75, 1905-1910. 10.1021/ac034667q CCC: $25.00

© 2003 American Chemical Society Published on Web 09/19/2003

Figure 1. Schematic representation of (a) traditional injection method and (b) double-L injection method in cross-form microfluidic chips.

method with an integrated postcolumn reactor.29 However, as of today, a detailed numerical and experimental analysis of the lowleakage (double-L) injection technique is still lacking. The injection system on a microfluidic chip is one of the key elements in the sample-handling process, and its characteristics determine the overall quality of the separation. Therefore, if the microfluidic chip design is to be optimized, it is essential to develop a thorough understanding of the mechanisms governing electrokinetic manipulations, particularly those associated with discrete injections. Many discussions relating to cross-injection systems on microfluidic chips can be found in the published literature. Although, in the first injection run, such systems are capable of providing an ideally sized and orientated sample plug to the separation channel, sample leakage tends to occur from the upper and lower microchannels after several runs, and this phenomenon reduces the detection performance of the device.15 The current study develops a mathematic model to analyze the electrical potential contours, velocity vectors, and streamline in a microfabricated CE chip and experimentally tests the double-L injection technique, which uses electrokinetic manipulations to prevent sample leakage. Schematic views of the traditional cross-injection system and the proposed double-L injection system are presented in Figure 1, in which the operating principles of the proposed technique are clearly shown in Figure 1b. This paper provides a detailed description of the proposed low-leakage injection technique and adopts experimental and numerical approaches to evaluate its performance when applied within the cross-form micro CE chip. It will be shown that the low leakage associated with the double-L injection technique improves the detection performance significantly and therefore renders the injection technique particularly suitable for use in applications where a highly sensitive sample separation is required. EXPERIMENTAL SECTION Fabrication Process. The microchip substrates used in the current experimental study were formed by cutting polished soda lime glasses for SDN-LCD displays (300 × 400 × 1.1 mm3, G-Tech Opto-electronics Corp., Taiwan) into plates measuring 30 mm × 80 mm. Figure 2 presents a simplified overview of the fabrication process that was used to form the planar microfluidic channels on the glass substrates.30 Initially, the glass substrates were (29) Jacobsen, S. C.; Koutny, L. B.; Hergenroder, R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472-3476. (30) Lin, C. H.; Lee, G. B.; Lin, Y. H.; Chang, G. L. J. Micromech. Microeng. 2001, 11, 726-732.

Figure 2. Fabrication of glass-based microfluidic chip.

Figure 3. (a) Photograph of a traditional cross-form micro CE chip and (b) SEM images of a closeup view for the etched microchannel.

cleaned thoroughly by placing them in a boiling piranha solution (H2SO4 (98%):H2O2 (30%) ) 3:1, volume ratio) for 10 min. Rather than using a time-consuming vacuum deposition process to fabricate the mask required to etch the plates in an HF-based etchant, this study applied a 3-µm-thick AZ4620 (Clariant Corp.) photoresist layer as the masking material in the wet chemical etching process. As shown in Figure 2a, a standard lithography process was used to generate the required configuration of the microchannels. Note that the width of these microchannels was 100 µm. The patterned substrates were then etched for 45 min in a 6:1 buffered oxide etchant (BOE; J. T. Baker) bath, which was agitated ultrasonically, to form 40-µm-deep microfluidic trenches (Figure 2b). As shown in Figure 2c, the PR layer was then removed. Meanwhile, holes were drilled in simple 30 mm × 80 mm glass plates and the plates cleaned in a boiling piranha solution (Figure 2d). The two glass flats were then carefully aligned and made to cling to each other using DI water, as shown in Figure 2e, and finally the two plates were thermally bonded in a sintering oven at 580 °C for 10 min (Figure 2f). Figure 3 presents photographs of the microchannel configurations considered in the present study. Figure 3a shows the crossform configuration used to provide standard-sized samples to the Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

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separation channel. Note that the microchannels measure 100 µm in width by 40 µm in depth. Figure 3b shows SEM images of the cross-form injection microchannels following the wet chemical etching process. The results confirm the effectiveness of the thin positive photoresist layer as an etching mask when glass etching in a BOE solution. Furthermore, the success of the developed fabrication process in forming microfluidic chips with well-defined patterns and highly smooth surfaces is clearly evident in the image. Experimental Procedure. The detection performance of the cross-form injection systems was tested experimentally with and without application of the proposed double-L injection technique. The injection step was driven by an applied voltage of 0.4 kV over a 0.5-min loading time, while the applied voltage during the separation step was established within the range of 0.6-1.5 kV over a 0.8-min separation time. The buffer solution used throughout this study was 1 mM Na2B4O7‚10H2O with a pH value of 9.2. A standard laser-induced fluorescence detection system comprising a fluorescence microscope (E-400, Nikon) and a mercury lamp module was used for the excitation and observation of the fluorescence dye. A CCD (model TE/CCD512TKM, CFMDIO, Taiwan) was used to acquire the optical images. Note that the concentration of the fluorescence dye (Rhodamine B) was adjusted to 3 × 10-4 M in order to improve the quality of the captured images. Finally, an APD module (Avalanche Photo-Diode, C5460-01, Hamamatsu) was used to detect the emitted optical signals. MATHEMATICAL MODEL Regarding the numerical simulation of electroosmotic flows, most previous studies assumed the velocity profile to be fully developed in the microchannels and considered the charge density to conform to the Boltzmann equilibrium distribution.31-35 However, these assumptions are not strictly valid since electroosmotic flow involves an entrance region within which neither condition is satisfied. The current authors have previously developed physical models based on (a) the Poisson equation for the electrical potential and ζ potential, (b) the Nernst-Planck equations for the ionic concentration, (c) the full Navier-Stokes equations modified to include the effects of the body force due to the electrical and charge densities,36-38 and (d) a concentration equation for the sample plug distribution. The physical model developed in the present study combines the Poisson equation for the applied electrical potential and the ζ potential of the fluid-solid boundary, the ionic concentration (n+, n-) for the positive and negative ions of the fluid, and the full Navier-Stokes equations modified to include electrokinetic force effects. By introducing the reference quantities Lref., Uref, pref, and Fref, and defining Fje ) Fe/n0ze ) (n+ - n-)/n0, ψ h ) zeψ/kbT, ht ) tUref/Lref, uj ) u/Uref, vj ) v/Uref, jx ) x/Lref, jy ) y/Lref, pj ) (p (31) Patankar, N. A.; Hu, H. H. Anal. Chem. 1998, 70, 1870-1881. (32) Griffiths, S.; Nilson, R. H. Anal. Chem. 2001, 73, 272-278. (33) Dutta, P.; Beskok, A. Anal. Chem. 2001, 73, 1979-1986. (34) Otevrˇel, M.; Klepa´rnı´k, K. Electrophoresis 2002, 23, 3572-3582. (35) Erickson, D.; Li, D. Langmuir 2002, 18, 1883-1892. (36) Fu, L. M.; Yang, R. J.; Lee, G. B. Electrophoresis 2002, 23, 602-612. (37) Lin, J. Y.; Fu, L. M.; Yang, R. J. J. Micromech. Microeng. 2002, 12, 955961. (38) Fu, L. M.; Lin, J. Y.; Yang, R. J. J. Colloid Interface Sci. 2003, 258, 266275.

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pref)/(FUref2), Fj ) Ff/Fref, nj + ) (n+ - n0)/n0, and nj - ) (n- - n0)/ n0, the dimensionless form of the governing equations after dropping the head symbols can be written as

κ2 ∂2ψ ∂2ψ + ) F 2 e ∂x2 ∂y2 ∂n( ∂n( ∂n( +u +v ) ∂t ∂x ∂y

(

)

(1)

[(

)] (2)

1 ∂2n( ∂2n( 1 ∂ (∂ψ ∂ ( ∂ψ + 2 ( n + n ScRe ∂x2 ScRe ∂x ∂x ∂y ∂y ∂y

)

(

∂u ∂v + )0 ∂x ∂y

( (

(3)

)

∂u ∂u ∂p 1 ∂2u ∂2u ∂ψ ∂u +u +v )+ + - GxFe ∂t ∂x ∂y ∂x Re ∂x2 ∂y2 ∂x

(4)

)

(5)

1 ∂C + u‚∇C ) ∇ 2C ∂t ScRe

(6)

∂v ∂v ∂v ∂p 1 ∂2v ∂2v ∂ψ + - GxFe +u +v )+ ∂t ∂x ∂y ∂y Re ∂x2 ∂y2 ∂y

In the equations above, κ ) WK, where K is the Debye-Huckel parameter and is given by K ) (2n0z2e2/0kbT)1/2, 1/K is the characteristic thickness of the charge density, Fe ) (n+ - n-)ze is the charge density, n+ and n- are the respective concentrations of the positive and negative ions,  is the dielectric constant of the medium, 0 is the permittivity of a vacuum, n0 is the bulk concentration of the ions, kb is the Boltzmann constant, T is the absolute temperature, Lref ) W, where W is the channel height, Uref ) ψinlet0|ζ|/µL, where ψinlet is the activated potential at the inlet, Re is the Reynolds numbers and is given by Re ) FfUrefLref/µ ) Ff(ψinlet0|ζ|/µL)(Lref/µ), Sc is the Schmidt number and is given by Sc ) µ/FfDi, µ is the liquid viscosity, Ff is the fluid density, Di is the diffusion coefficient of the sample, ζ is the surface zeta potential, p is the pressure, Gx is the ratio of the EDL energy to the mechanical kinetic energy and is given by Gx ) 2n0kbTFfW2/ µRe2, and finally, C is the sample concentration. The more detailed expressions of the numerical technique are given in the Supporting Information. The current study uses microchips made of silica glass substrates and sodium borate as the buffer fluid. The dielectric constant, electrokinetic diameter, channel wall ζ potential, buffer fluid viscosity, charge density Schmidt number, and sample diffusion coefficient are given respectively by the following:

[ ]  ∼ 80 κ ∼ 32 ζ ∼ -75 mV µ ∼ 10 - 3 N ‚s/m2 Sc ∼ 105 D ∼ 6.9 × 10 - 11m/s

(7)

RESULTS AND DISCUSSION Traditional Cross-Form Injection Method. Figure 1a presents the traditional cross-form injection channel. Many previous

Table 1

experimental investigations into capillary electrophoresis have adopted this particular injection channel configuration and have shown how the sample may be driven into the separation channel by means of an appropriate manipulation of the applied voltage. Although the injection and separation channels are mutually perpendicular, it is noted that the overall configuration of the microfluidic device may not be quite so simple. Injection of the sample involves discrete injection and separation steps, during

which the controlling electrical fields are established in accordance with Kirchhoff’s law.14 In a previous study, the current authors have described the potential fields required to control the sample flow in the injection and separation steps in a cross-form microfluidic device.27 Table 1 presents the electrical potential strengths applied during the current experimental and numerical investigations of the traditional cross-form injection microfluidic device. The shape of the delivered sample plug depends primarily on the electroosmotic flow pattern at the intersection of the channels and plays an important role in determining the resolution of the electrophoresis analysis. Figure 4 presents the electrical potential contours, velocity vectors, and streamlines that are established during the separation step in the cross-form injection method. Channels 1 and 2 are opened, and the values of φ1 and φ2 are both larger than φ4. Therefore, although potential gradients exist in channels 1 f 4 and 2 f 4, they are smaller than the potential gradient in channel 3 f 4 (Figure 4a). Observation of the velocity vectors and streamlines in Figures 4b and c shows clearly that sample leakage takes place from channels 1 and 2 in this particular injection method. Figure 5 compares the experimentally obtained CCD images and the numerically simulated results of the continuous sample injection distribution. The two images shown in Figure 5a show the flow of the sample from the sample inlet channel to the waste channel under the influence of an injection voltage of 0.4 kV. When

Figure 4. (a) Electrical potential contours, (b) velocity vectors, and (c) streamline plots of separation step in traditional cross-form injection method.

Figure 5. Comparison of experimental and simulated results of sample distributions in traditional cross-form injection method: (a) injection step, (b) separation step with voltage of 0.6 kV, and (c) separation step with voltage of 1.5 kV.

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Figure 6. Acquired optical signals for a series of more than 10 injection/separation cycles in traditional cross-form injection method with separation voltages of (a) 0.6, (b) 0.9, (c) 1.2, and (c) 1.5 kV.

Figure 7. Comparison of the sample leakage ratio under various separation voltages.

Figure 8. (a) Electrical potential contours and (c) streamline plots of separation step in modified cross-form injection method.

the sample has completely filled the cross-channel intersection, the electric potential switches immediately to the separation step, in which channels 1 and 2 are maintained at open (i.e., ∂φ/∂x ) 0) and a separation voltage of 0.6 kV is applied. Under these conditions, the sample is forced out of the intersection into the separation channel, as shown in Figure 5b. At this level of separation voltage, a low leakage of the sample plug from channels 1 and 2 is noted as the plug is injected into the separation channel. However, as shown in Figure 5c, when the separation voltage is 5794

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increased to 1.5 kV, sample leakage from channels 1 and 2 becomes very evident and will cause a deterioration in the resolution of the separated components within the sample. To understand the effects of sample leakage on the resolution sensitivity of the cross-form microchip, a constant injection voltage of 0.4 kV was established, and a series of injection/separation cycles was performed at various levels of separation voltage. A test sample of 3 × 10-4 M Rhodamine B was used in each case. The sample was excited within the detection region using a mercury lamp, and an APD module was then used to detect the resulting optical signals. Panels a-d of Figure 6 present the acquired optical signals over a series of more than 10 injection/ separation cycles performed at separation voltages of 0.6, 0.9, 1.2, and 1.5 kV, respectively. The figures indicate that the baseline begins to drift as the number of completed injection/separation cycles increases. This drift effect is the result of sample leakage from channels 1 and 2. It is very obvious that the drift effect becomes more significant as the separation voltage increases; i.e., it becomes evident after 11 cycles for a low separation voltage of 0.6 kV but is noticeable after just one cycle at the highest voltage of 1.5 kV. Figure 7 presents the sample leakage ratio for the conventional cross-form and double-L injection method in the separation channel generated with different applied voltages. The average sample leakage ratio was evaluated by measuring the leakage area across the microchannel for three times during the experiment. The variation of the measurement was within 6%. A linear relationship is seen to exist between the leakage (%, volume ratio) and the separation voltage under 1.5 kV. Meanwhile, the double-L injection technique provided a very small leakage ratio in compare with the conventional cross-form injection method. Modified Cross-Form Injection Method. In the traditional cross-form injection method described above, channels 1 and 2 are maintained at open during the separation step, and therefore, fluid flow takes place within them, as shown in Figure 4c. As demonstrated in Figure 5, this results in sample leakage, and therefore, it is necessary to apply a low voltage to the sample inlet and waste channel reservoirs during the separation process. This causes the sample fluids within these channels to flow to their

Figure 9. Continuous sample injection distributions in separation step of modified cross-form injection method with separation voltage of 1.2 kV.

Figure 10. Electrical potential contours in double-L injection method: (a) injection and (b) separation steps with separation voltage of 1.2 kV.

respective reservoirs as the sample plug is driven out of the intersection into the separation channel under the influence of the applied separation voltage. Consequently, the leakage effect is minimized and the resolution performance is improved. (Note that the corresponding applied voltages for this modified crossform injection method are presented in the second row of Table 1). Figure 8 presents the electrical potential contours and the velocity streamlines of the separation step in the modified crossform injection method. In Figure 8a, it can be seen that the electrical potentials of the reservoirs in channels 1 and 2 are smaller than the potential in the intersection area. Consequently, the velocity streamlines shown in Figure 8b flow predominantly from channel 3 to channel 4, with just a minority guided into channels 1 and 2. Figure 9 presents the numerically simulated results for the distribution of the continuous sample injection in the modified cross-form injection method for an applied separation voltage of 1.2 kV. Once the sample has completely filled the intersection between the injection channels (channels 1 and 2) and the separation channels (channels 3 and 4), the electrical potential switches to the separation step. The sample plug is led into the separation channel, and meanwhile, the sample fluid in channels 1 and 2 is driven into the reservoirs of the injection channels. Although it can be seen that this technique is successful in reducing the sample leakage from channels 1 and 2, it requires the electrical potential to be controlled at four different points during the separation step (i.e., φ1, φ2, φ3, and φ4). Clearly,

Figure 11. Comparison of experimental and numerical simulation results for sample distributions in double-L injection method: (a) injection step with voltage of 0.4 kV and (b) separation step with voltage of 1.5 kV.

therefore, this modified injection method is more complex than the traditional cross-form injection method, in which only two control points are required, i.e., φ3 and φ4. Double-L Injection Method in Cross-Form Microchip. To overcome the problems of flow leakage without resorting to the complex potential control required by the modified cross-form injection method, the present study proposes the use of a novel double-L injection method, as shown in Figure 1b. Figure 10 presents the electrical potential contours and streamlines during the injection and separation steps of the proposed double-L injection method. During the injection step, the sample is loaded from channel 1 to channel 3, and the buffer flows in channels 2 and 4 are prevented from moving. Therefore, as shown in Figure 10a, the electrical potentials in channels 1 and 3 must generate a driving force (i.e., ∂φ/∂S * 0, where S indicates the injecting path) to load the sample, while the electrical potentials in channels 2 and 4 must be maintained at a constant value (i.e., ∂φ/∂S ) 0; i.e., opened). During the separation step, the sample in the intersection is led into the separation channel (i.e., channel 4), and the electric potentials in channels 1 and 3 are maintained at a constant value (i.e., opened, but φ3 > φ1). The streamline flows in Figure 10b clearly show that, during the separation step, the fluid flows predominantly from channel 2 to channel 4, while a very small flow is pushed toward the reservoirs of channels 1 and 3. Note that the pressure drop in the (39) Yang, R. J.; Fu, L. M.; Lin, Y. C., J. Colloid Interface Sci. 2001, 239, 98105.

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that the double-L injection method is successfully able to minimize sample leakage and is therefore appropriate for use in separation applications requiring a high resolution.

Figure 12. Acquired optical signals for series of 15 injection/ separation cycles of 3 × 10-4 M Rhodamine B in double-L injection method with injection voltage of 0.4 kV and separation voltage of 1.5 kV.

microchannel utilizing electrokinetic pumping is very small and has extremely little effect on the simulation and experimental results. The detailed description can be found in our previous work.39 Figure 11 compares the simulation results and the experimental observations of the sample distribution during the injection and separation steps of the proposed double-L injection method. Figure 11b shows a clear reduction in the sample leakage into channel 4 when this injection method is applied. Figure 12 shows the acquired optical signals obtained for the double-L injection method for a series of 15 injection/separation cycles of a 3 × 10-4 M Rhodamine B sample at an injection voltage of 0.4 kV and a separation voltage of 1.5 kV. Compared with the results presented in Figure 6d for the traditional cross-form injection system, it is noted that, in this case, the baseline over the 15 cycles remains very stable. Therefore, it can be concluded

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CONCLUSIONS This paper has presented a numerical and experimental investigation into low-leakage injection methods for microfluidic chips. The mathematical model and its computer implementation have demonstrated that computer simulation is a useful tool in representing the injection process of a sample into a separation channel. The current study has identified and characterized sample leakage in the separation channel and has suggested that this is the cause of the baseline drift observed in the optical signals acquired over a series of routine microfluidic chip-based capillary electrophoresis separations. It has been noted that the sample leakage associated with the traditional cross-form injection method tends to degrade the detection performance of these microfluidic devices. This study has proposed a novel double-L injection method to minimize sample leakage and has investigated its performance experimentally and numerically when applied to the traditional cross-form injection method. The results have shown that the proposed method is effective in minimizing sample leakage and have demonstrated its suitability for use in applications requiring a high degree of detection sensitivity. It is the current authors’ firm belief that the proposed double-L injection method represents an ideal basis for the development of the next generation of microfluidic devices. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 20, 2003. Accepted August 19, 2003. AC034667Q