Anal. Chem. 1995, 67,2059-2063
Fused Quartz Substrates for Microchip Electrophoresis Stephen C. Jacobson,* Ahrln W. Moore, and J. Mlchael Ramrey Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, P.0. Box 2008 Oak Ridge, Tennessee 37831-6142
Afused quartz microchip is fabricated to perform capiUary electrophoresis of metal ions complexed with B-hydroxyquinoline-5-sulfodcacid (HQS). The channel manifold on the quartz substrate is fabricated using standard photolithographic, etching, and depositiontechniques.By incorporating a direct bonding technique during the fabrication ofthe microchip,the substrateand m e r plate can be fused together below the melting temperature for fused quartz. To enhance the resolution for the separation, the electroosmoticflow is “klby covalently bonding polyacryiamide to the channel walls. A separation length of 16.5 m m and separation field strength of 870 V/cm enable separationsto be performed in 5 15 s. By increasingthe concentrationof HQS from 5 mM to 20 mM, the separation ef6ciency improves by -3 times. The low background signal from the fused quartz substrate results in mass detection limits of 85,61, and 134 am01 and Concentration detectionlimits of 46,57, A d 30 ppb for Zn,Cd, and Al, respectively. Miniaturized chemical analysis instruments fabricated using micromachining techniques are receiving increasing attention. The more successful demonstrations for miniaturized instruments incorporate liquid phase separation techniques. Devices have been demonstrated for capillary electrophoresis,’-6 freeflow electrophoresis,7open channel electrochromatographyraphy,8and capillary electrophoresis with pre and postcolumn derivatization?JO All of the previously demonstrated devices utilize glass substrates. By moving to quartz substrates, the superior optical properties of quartz especially in the W region can be exploited. For both capillary electrophoresis and liquid chromatography, the most useful wavelength range for the purpose of optical detection is 200-400 nm, and the ability to fabricate fused quartz microchips permits investigation of similar detection scenarios. (1) Harrison, D. J.; Manz, A; Fan,Z.; Liidi, H.; Widmer, H. M. Anal. Chem. 1992,64, 1926. (2) Manz, A; Harrison, J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A; Liidi, H.; Widmer, H. M. J Chromatop. 1992,593, 253. (3) Seiler, IC; Harrison, D. J.; Manz, A Anal. Chem. 1993,65, 1481. (4) Harrison, D. J.; Run, IC; Seiler, IC; Fan,Z.; Effenhauser, C. S.; Manz, A Science 1993,261, 895. (5) Jacobson,S. C.;Hergenroder, R; Koutny, L. B.; Warmack, R J.; Ramsey,J. M. Anal. Chem. 1994,66,1107. (6) Jacobson, S. C.; Hergenroder, R; Koutny, L.B.; Ramsey, J. M. Anal. Chem. 1994,66, 1114. (7)Raymond, D. E.; Manz, A; Widmer, H. M. Anal. Chem. 1994,66, 2858. (8)Jacobson, S. C.; Hergenroder, R; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994,66, 2369. (9) Jacobson, S. C.; Hergenroder, R; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994,66,4127. (10) Jacobson,S. C.; Koutny, L B.; Hergenroder, R; Moore, A. W., Jr.;Ramsey, J. M. Anal. Chem. 1994,66, 3472.
0003-2700/95/0367-2059$9.00/0 Q 1995 American Chemical Society
injection
sample reservoir separation
cross
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buffer reservoir
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Figure 1. Schematic of the fused quartz microchip. The reservoirs are affixed on the microchip via epoxy.
To test the performance of fused quartz substrates, metal ions complexed with 8-hydroxyquinoline5sulfonicacid (HQS) are separated by electrophoresis and detected with W laser-induced fluorescence. HQS has been widely used as a ligand for optical determinations of metal ions following initial work by Fiegl and Heisig.I1 The optical properties and the solubility of HQS in aqueous media have recently been used for detection of metal ions separated by ion chromatography12and capillary electrophoresi~.’~Because uncomplexed HQS does not fluoresce, excess ligand is added to the buffer to maintain the complexation equilibria during the separation without contributing a large background signal. This benefits both the efficiency of the separation and the detectability of the sample. In this paper, the fabrication of fused quartz microchips is described. Direct bonding of the cover plate to the substrate enables covalent bonding to be performed below the melting temperature of fused quartz. A simple cross channel manifold ( F i r e 1) is constructed to perform electrophoretic analysis of metal ions complexed with HQS. The electroosmoticflow of the microchip is minimized by covalently bonding polyacrylamide to the surface of the channels. This enables a higher resolution to be achieved in a shorter separation time and length. Improved efficiency with increasing HQS concentration is also discussed. EXPERIMENTAL SECTION
The microchips are fabricated using standard photolithographic, wet chemical etching and bonding techniques.14 First, (11) Fiegl, F.; Heisig, G. B. Anal. Chim. Acta 1949,3,561. (12) Soroka, IC; Vithanage, R S.; Phillips, D. A; Walker, B.; Dasgupta, P. IC Anal. Chem. 1987,59, 629. (13) Swaile, D.F.; Sepaniak, M. J. Anal. Chem. 1991,63,179. (14) For example: KO, W. H.; Suminto, J. T. In Sensors; Gopel, W., Hasse, J., Zemmel, J. N., Eds.; VCH: Weinhein, Germany, 1989; Vol. 1, pp 107-168.
Analytical Chemistry, Vol. 67, No. 13, July 1 , 1995 2059
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width [pm] Figure 2. Profile of separation channel machined on a fused quartz substrate.
the fused quartz substrates (50 mm x 25 mm x 1 mm; Esco Products, Inc. R120110) are sputtered with metal films of chromium (30 nm) and gold (400 nm) . A positive photoresist (Shipley 1811) is then spincoated on top of the Au film, and the column design (Figure 1) is transferred to the substrate using an e-beam written photomask (Institute of Advanced ManSciences, Inc.). Following exposure, the metal films are etched using KI/ IZfor Au and &Fe(CN)G/NaOH for Cr. Finally, the channels are etched into the substrate in a dilute, stirred HF/NH4F bath at 50 "C. Due to the isotropic etch of amorphous materials, the channel profile is trapezoidal. Figure 2 shows the cross section of the quartz channel measured by a profilometer (Alpha Step 200, Tencor Instruments) prior to bonding the cover plate. The channel depth is 7.6 pm, and the channel width at halfdepth is 75 pm. To form the closed network of channels, a circular cover plate (25 mm diameter; Esco Products, Inc. R525000) is bonded to the substrate over the etched channels. The substrate and cover plate are fust hydrolyzed in NH40H/H202 at 50 "C, rinsed in HzO, joined, and then annealed at 1100 "C for 5 h. Cylindrical glass reservoirs are affixed on the substrate using epoxy. The channel walls are then coated with polyacrylamide to m i n i i e electroosmotic fl0w.15 First, the channels of the microchip are filled with a solution of [ y(methacryloxy)propylltrimethoxysilane 0.4%(v/v) in water (PH 3.5, adjusted with acetic acid) for 30 min. The channels are then flushed with water and filled with a 3% (w/v) solution of acrylamide in water with 0.1% (v/v) Nfl,N',N'tetramethylethylenediamine and 0.1%(w/v) potassium persulfate for 30 min. The excess acrylamide solution is removed, and the channels are rinsed sequentiallywith water and separation buffer. Column performance and separations are monitored onmicrochip using a single-point detection scheme via laser-induced fluorescence &IF). An argon ion laser (351.1-363.8 nm, 10 mW Coherent Innova 90) is used for excitation and focused to a spot on the microchip using a lens (100 mm focal length). The fluorescence signal is collected using a 21x microscope objective, followed by spatial fltering (0.6 mm diameter pinhole) and spectral filtering (425 nm cutofl; Coming 3-73), and measured using a photomultiplier tube (PMT; Oriel 77340). The data acquisition/ voltage switching apparatus is computer controlled using programs written in-house in Labview 3.0 (National Instruments). Platinum electrodes provide electrical contact from the power supply (Spellman CZElOOOR) to the solutions in the reservoirs. The compounds used for the experiments are zinc sulfate, cadmium nitrate, and aluminum nitrate. The buffer is sodium phosphate (60 mM, pH 6.9) with &hydroxyquinoline-5sulfonic acid (20 mM for all experiments except that in Figure 5; Sigma Chemical Co.). At least 50 mM sodium phosphate buffer is needed to dissolve up to 20 mM HQS. (15) Hjerten, S.J. Chromatogr. 1985, 347, 191. 2060 Analytical Chemistry, Vol. 67, No. 73, July 7, 1995
5
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Figure 3. Electropherogram of Zn, Cd, and AI complexed with HQS: (a, top) Esep = 870 V/cm and Lsep= 16.5 mm. The injected concentrations are 8.5 (130), 6.7 (60), and 4.3 ppm (160pM) for Zn, Cd, and AI, respectively (b, bottom) Esep = 720 V/cm and Lsep = 16.5 mm. The injected concentrations are 260 (4.0), 210 (1.9), and 130 ppb (4.9 pM) for Zn, Cd, and AI, respectively.
The microchip is operated in either a sample loading or separation mode. The floating sample loading, described previously,4 is used to transport the sample to the injection cross, enabling a larger volume sample plug to be injected onto the separation column than with the pinched sample loading. The average sample plug volume for the floating sample loading is estimated to be 120 pL compared to 65 pL for the pinched sample loading. These volumes are determined by measuring injected plug widths 0.1 mm downstream from the injection cross. For comparison, the volume of the injection cross is 43 pL. With the floating sample loading, the injected plug has no electrophoretic bias, but the volume of sample is a function of the sample loading time. Because the sample loading time is inversely proportional to the field strength used, for high sample loading field strengths a shorter sample loading time is used than for low injection field strengths. For example, for a sample loading field strength of 630 V/cm ( F i r e 3a), the sample loading time is 12 s, and for a sample loading field strength of 520 V/cm (Figure 3b), the sample loading time is 14.5 s. Both the pinched and floating sample loadings can be used with and without suppression of the electroosmotic flow. To implement the floating sample loading,
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Table 1. Data for Figure 3a
metal ion
electro horetic
mobility (l$cm2 V-l
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plate no.
Zn
1.65
8200
Cd
1.49
6500
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2.26
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2.86
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3200
RESULTS AND DISCUSSION
Figure 3 shows the separation of three metal ions complexed with 8hydroxyquinoline5sulfonic acid. All three complexes have a net negative charge. With the electroosmotic flow minimized by the covalent bonding of polyacrylamide to the channel walls, negative potentials relative to ground are used to manipulate the complexes during sample loading and separation. In panels a and b of Figure 3, the separation channel field strength is 870 and 720 V/cm, respectively, and the separation length is 16.5 mm. The volume of the injection plug is 120 pL, which corresponds to 16, 7, and 19 fmol injected for Zn, Cd, and Al, respectively, for Figure 3a. In Figure 3b, 0.48, 0.23, and 0.59 fmol of Zn, Cd, and Al, respectively, are injected onto the separation column. The average reproducibility of the amounts injected is 1.6%relative standard deviation (rsd) as measured by peak areas (six replicate analyses). The stability of the laser used to excite the complexes is -1% rsd. The primary purpose of eliminating the electroosmotic flow is to enhance the resolution of the separation. For electrophoretic separations, the resolution between two components can be written16
where N is the average number of theoretical plates, p.4 and p~ are the electrophoretic mobilities for components A and B, respectively, ~ A isV the average electrophoretic mobility for A and B, and p ~ ois the electroosmotic mobility. Electroosmosis does not contribute to the separation. To maximize the resolution, electroosmosis must be eliminated or precisely controlled, e.g., application of an external field.17 For simplicity, electroosmosis is minimized by covalently bonding polyacrylamide to the channel surface. Table 1 lists the efficiencies and resolution (eq 1) generated for each complex in Figure 3a. The resolution obtained between (16)Jorgenson, J. W.; Lukacs, K D. Anal. Chem. 1981,53, 1298. (17) Lee, C. S.; Banchard, W. C.; Wu, C. T.Anal. Chem. 1990, 62, 1550.
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a voltage is applied to the sample reservoir with the sample waste reservoir grounded and the buffer and waste reservoirs floating. After the slowest moving ion, e.g., Al, passes through the injection cross, the applied potentials are recodgured for the separation mode. In the separation mode, the voltage is applied to the buffer reservoir with the waste reservoir grounded and with sample and sample waste reservoirs at 60%of the buffer reservoir potential. Having a voltage applied to the sample and sample waste reservoirs at a fraction of the buffer reservoir voltage prevents excess sample from bleeding into the separation column.
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Zn/Cd and Cd/Al is 2.25 and 2.86, respectively. To obtain a comparable resolution, e.g., 2.25, with electroosmotic flow would require a longer separation column and analysis time. For example, with an electroosmotic mobility of 5.7 x cm2V-' s-1 (measured on a microchip without the electroosmotic flow minimized), a field strength of 870 V/cm and a resolution of 2.25, a separation length of 11.5 cm is required to resolve these three metal ion complexes, and the total analysis time increases to 33 s. Also, to produce the equivalent separation field strength, a 9.9 kV power supply is required compared to 1.4 kV for this separation. The smaller power supply is more amenable to miniaturization. The analysis time can be reduced by increasing the separation field strength and, consequently, the linear velocity of the sample16 24
= PEsep
(2)
where p is the electrophoretic mobility and Esepis the separation field strength. The electrophoretic mobility depends on the viscosity of the buffer. If the heat generated in the separation column is not dissipated efficiently during a separation, the viscosity of the buffer decreases, leading to higher than predicted linear velocities for the sample zones. In Figure 4 the linear velocity of the sample zones are plotted versus the electric field strength. For separation field strengths of L 720 V/cm, a deviation from linear behavior is observed. Also, plotted in Figure 4 is the current measured in the separation column. As expected, the current in the separation column exhibits the same nonlinear behavior for field strengths of 2720 V/cm. For separation field strengths of 720 and 870 V/cm, the microchip required -2 min for the linear velocities to stabilize. The reproducibility of the linear velocities is 0.1%rsd (six replicate analyses). To reduce this heating effect at comparable separation field strengths, channels with smaller cross-sectionalTeas or lower concentration (lower conductivity) buffers are two possibilities. Optimum HQS concentration for detection and separation (discussed below) is determined to be 20 mM, which requires at least 50 mM phosphate buffer for dissolution. Thus, 80 mM for a total buffer concentration is near the operating limit for these experiments. Analytical Chemistry, Vol. 67,No. 13, July 1, 1995
2061
Table 2. Detection Limits
concentration metal ion
PM
ppb
massa (mol)
Zn Cd
0.71 0.51 1.12
46 57 30
85 61 134
Al a
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dominant form for Zn and Cd is a 2:l ligand-metal complex, and for Al a 3:l complex. The Al complex with a 3- charge might undergo an ion-pairing phenomena. If two or more species exist in the sample band for Al, additional band dispersion results because the species will migrate at d ~ e r e nvelocities, t yet cannot be resolved under these conditions. For HQS concentrations of '20 mM, signilicant improvements in the efficiency are not observed. The relationships between HQS concentration, the complex formation constant, and band broadening associated with HQS-metal complexes in capillary electrophoresis have been investigated.lZ Additional band broadening was previously attributed to nonequilibrium conditions at injection, various' HQS metal species in the sample band, and competing equilibria with supporting electrolyte.1z For these experiments, nonequilibrium conditions at injection do not occur because the sample and separation buffers are the same. With the magnitude of the formation constants listed above and the large excess of HQS, multiple metal-HQS species seems improbable. For electrophoresis, the contributions to the total plate height from the injection plug length, detector observation length,20axial and Joule heatingzzare
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HQS concentration [mM] Figure 5. Variation of plate height for Zn (0),Cd (O), and AI (0) with concentration of HQS. Error bars are &a for three analyses.
In addition to resolving power and analysis time, detection must also be considered in an analytical procedure. Detection limits are measured using serial dilutions of a stock solution of the metal ions. The measurements are performed by separating the mixture of the metal ions, using the experimental conditions in F i r e 3b. The data are then extrapolated to a signal-twoise ratio (S/ N) of 2, to give the concentration and mass detection limitslisted in Table 2. Figure 3b shows the electropherogram at the lowest concentration injected for the mixture during the detection limit study. The peaks correspond to an average S/N for 3 runs of 16.4,10.7,and 8.6 for Zn, Cd, and Al, respectively, which are within 1 order of magnitude of the estimated detection limits Vable 2). A pH 6.9 separation buffer is near the optimum conditions for fluorescence detection of the complexes with Zn, Cd, and Al." Detection limits are improved by using a fused quartz substrate as opposed to a glass substrate. The observed background signal from the experimental apparatus with the fused quartz substrate inserted is 30%of the background signal observed for glass. The primary contributionsto the background signal include stray room light, scattered laser light, and inelastic scattering from the microscope objective and microchip substrate. Further reductions in background signals are expected by improving the quality of the optical components. A substantial decrease in the plate height is observed with increasing concentrationsof HQS in the separation buffer. Figure 5 shows the variation of the plate height with concentration of the HQS. The Al complex exhibits the poorest efficiency. The and W , 1 9 formation constants for Zn, Cd, and Al are 1016,z,1014.2,18 respectively. Because of the large formation constants, the (18) Lange's Handbook of Chemkty, 14th ed.; Dean,J. A, Ed.; McGraw-Hill Book Co.: New York, 1992.
2062 Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
where l,, is the injection plug length, ldet is the detector observation length, LP is the separation length, Dmis the diffusion coefficient, d, is the channel 1 is the molar conductivity of the buffer, Cis the buffer concentration, and K is the thermal conductivity of the buffer. The contributions from the injection plug length and detector observation length assume Gaussian distributions. The lengths of the injection plug and the detector observation are constant for all experiments. If these heindependent contributions predominate in their contribution to the plate height, then the total plate height decreases as the separation length increases. The contributionfrom axial diffusion to the plate height is reduced by increasing the separation field strength and, consequently, reducing the analysis time. The contribution from Joule heating can be minimized by controlling the channel dimensions, separation field strength, and buffer concentration. In Figure 6, the variation of the total plate height with the electric field strength is plotted. For these experiments, the injection plug length is 330 pm and the detector observation length is 80 pm. The corresponding contributions to the plate height from the injection plug length and detector observation length are 0.41 pm and 24 nm, respectively. These contributions are small relative to the measured plate heights. The contribution from axial diffusion decreases with increasing field strength. (19) Bishop, J. A Anal. Chem. Acta 1973,63, 305. (20) Stemberg, J. C. Adu. Chromatogr. 1966,2,205. (21) Giddmgs, J. C. Dynamics of Chromatography, Part I: Principles and Theoy; Marcel Dekker: New York, 1965; Chapter 2. (22) b o x , J. H.; Grant, I. H. Chromatographia 1987,24,135. (23) d, usually corresponds to the capillary diameter. In this case, a channel with a trapezoidal cross section is under consideration, and the channel depth will be used for estimating the contribution of Joule heating to the plate height. (24) Monnig, C. A; Jorgenson, J. W. Anal. Chem. 1991,63, 802. (25) Hjertkn, S.; Elenbring, IC;KilAr, F.; Liao, J.-L; Chen, A; Siebert, C. J.; Zhu, M.-D. J. Chromatogr. 1987,403, 47. (26) Aebersold, R; Morrison, H. D. J. Chromatogr. 1990,516, 79. (27) Jandik, P.; Jones, W. R. J Chromatogr. 1991,546,431.
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separation field strength [V/cm] Figure 6. Variation of plate height for Zn (0),Cd (O), and AI (0) and powedlength (A)with the separation field strength complexed with HQS. The solid line is the calculated plate height for Zn using eq 3. Error bars are fo for three analyses.
However, the experimental plate height data do not asymptotically approach the sum of the time-independent contributions at high field strengths. In Figure 6, the calculated plate height for Zn using eq 3 (uzn = 1.65 x cm2V-' s-', Dm = 5 x cm2 s-') is plotted. For separation field strengths of 5430 V/cm, the experimental data correspond well with the calculated values, but for separation field strengths of 2580 V/cm, the agreement is poor. This difference could be due to heating of the microchip, as observed in the nonlinear behavior of the linear velocities (Figure 4). The power/length in the separation column is also plotted in Figure 6 for the corresponding field strengths. QpicaUy for electrophoretic systems, when the power/length remains below 1 W/m,24 Joule heating does not interfere with the separation performance. For separation field strengths of 2 580 V/cm, the power/length is > 1W/m. Using the last term for Joule (28) Chien, R-L.; Burgi, D. S. J. Chromatogr. 1991,559, 141. (29)Chien, R.-L.; Burgi, D. S. J. Chromatogr. 1991,559, 153. (30) Jacobson, S. C.; Ramsey, J. M. Electrophoresis, in press.
heating in eq 3, this contribution can be estimated. For p (Zn) = 1.65 x lo-* cm2V-I s-I, Esep= 870 V/cm, d, = 7.6 pm, A = 0.015 m2 mol-' Q-', C = 80 mM, Dm = 5 x cm2 s-l, and K = 0.6 W m-' K-l; the contribution of Joule heating to the plate height is 6.5 pm. As calculated, this contribution is insignificant relative to the plate height data. An alternative explanation for this discrepancy in the experimental and calculated plate height might be due to geometrical fluctuations in the channel over the length of the separation channel. As fabrication techniques for glass and quartz substrates improve, this discrepancy is likely to decrease as was the case for silica capillaries. In conclusion, metal ions complexed with &hydroxyquinoline5sulfonic acid are resolved in less than 15 s using microchip electrophoresis, and detection limits are in the range of 30-60 ppb. To enhance the detection limits for these complexes, a preconcentration step can be added prior to the separation. A sample preconcentration method that can be easily incorporated with electrophoretic analyses is field-amplified sample injection and sample ~tacking.2~-28Enhancements in detection limits up to 103 times have been observed using stacking methods with conventional capillary electrophore~is.~~ Similar stacking procedures have been demonstrated on microchips30and may allow subppb detection limits for metal ions. ACKNOWLEDGMENT
This research is sponsored by the US.Department of Energy (DOE), Office of Research and Development. Oak Ridge National Laboratory is managed by Martin Marietta Energy Systems, Inc. for the U S . Department of Energy under Contract DEAC05 840R21400. Also, this research is sponsored in part by an appointment for AW.M. to the ORNL Postdoctoral Research Associates Program. These postdoctoral programs are administered by the Oak Ridge Institute for Science and Education and ORNL. Received for review December 13, 1994. Accepted April 10, 1995. AC9412037 ~
@Abstractpublished in Aduance ACS Abstracts, June 1, 1995.
Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
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