Anal. Chem. 1994,66, 4127-4132
Precolumn Reactions with Electrophoretic Analysis Integrated on a Microchip Stephen C. Jacobson, Roland Hergenruder, Alvin W. Moore, Jr., and J. Michael Ramsey* Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Building 4500S,MS 6142, Oak Ridge, Tennessee 37831-6142
A glass microchip was constructed to perform chemical reactions and capillary electrophoresissequentially. The channel manifold on the glass substrate was fabricated using standard photolithographic, etching, and deposition techniques. The microchip has a reaction chamber with a 1 nL reaction volume and a separation column with a 15.4 mm separation length. Electrical control of the buffer, analyte, and reagent streams made possible the precise manipulation of the fluids within the channel manifold. The microchipwas operated under a continuous reaction mode with gated injections to introduce the reaction product onto the separation column with high reproducibility (< 1.8%rsd in peak area). The reaction and separation performances were evaluated by reacting amino acids with o-phthaldialdehydeto generate a fluorescent product which was detected by laser-induced fluorescence. Control of the reaction and separation conditionswas sacient to measure reaction kinetics and variation of detection limits with reaction time. Ha-times of reaction of 5.1 and 6.2 s and detection limits of 0.55 and 0.83 fmol were measured for arginine and glycine, respectively.
The microfabrication of analytical instrumentation will potentially enable the laboratory to be transported to the samples rather than vice versa. Miniature chemical instrumentation can be based on conventional laboratory approaches to chemical measurement problems. Many laboratory-based procedures require sample manipulation prior to the actual measurement, and many analyses are fully automated to circumvent operator bias. Similarly, a portable instrument should have these sample preparatory steps incorporated into the design and function of the instrument. In addition, micromachined chemical instruments as opposed to single-analyte chemical sensors should be able to identify and quantify all members of a desired class of compounds using a single device. The approach taken is to micromachine a channel manifold in a monolithic device that includes all desired fluid manipulation for complete chemical analysis. Chemical separation techniques including capillary electrophoresis,1-6 free-flow elec(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 Chromatogr. 1992,593, 253. (3) Seiler, K; Harrison, D. J.; Manz, A .4nal. Chem. 1993,65, 1481. (4) Hamson, D. J.; Fluri, K; Seiler, K; 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. 0003-2700/94/0366-4127$04.50/0 0 1994 American Chemical Society
trophore~is,~ open channel electrochromatography,s and capillary electrophoresis with postcolumn derivati~ation~ have been demonstrated using microfabricated devices. To demonstrate a reactor/analyzer microchip, we chose the relatively simple reaction of amino acids with o-phthaldialdehyde (OPA), which yields a fluorescent product.'O For capillary electrophoresis, both pre- and postseparation labeling using OPA have been studied.ll The reaction is moderately fast (a half-time of reaction for alanine at room temperature of x4 s l2 ), but the fluorescent product can be short lived (x10 min for glycineI3 1. This reaction is fast enough to perform postcolumn labeling when the analysis time is long compared to the reaction time, but as analysis times decrease, the postcolumn labeling becomes less attractive. Both detection limits and separation efficiency suffer due to "slow" product formation when high-speed analysis is p e r f ~ r m e d .Analysis ~ times on microchip electrophoresis devices can be significantly faster than the OPA reaction times; separation times for microchip electrophoresis as short as 150 ms have been reported.'j Precolumn derivatization becomes the desired method if the electrophoretic analysis can be performed before the product degrades and without hindering the quality of the separation. In this paper, a simple channel manifold was fabricated on a glass microchip to perform on-line precolumn reactions coupled with electrophoretic analysis of the reaction products. Here, the reactor is operated continuously with small aliquots introduced periodically onto the separation column to be analyzed. The operation of the microchip consists of three elements: derivatization of the amino acids with OPA, injection of the sample onto the separation column, and separationldetection of the components of the reactor effluent. Each of these elements is evaluated. EXPERIMENTAL SECTION
The microchips were fabricated using standard photolithographic, wet chemical etching and bonding techniques.14 A (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.; Koutny, L. B.; Hergenroder, R; Moore, A. W., Jr., Ramsey, J. M. Anal. Chem. 1994,66,3472. (10) Roth, M. Anal. Chem. 1971,43, 880.
(11) For example: Rose, D. J.; Jorgenson, J. W,J Chromatogr. 1988,447,117. Pentoney, S. L., Jr.; Huang, X.; Burgi, D. S.; Zare, R N. Anal. Chem. 1988, 60, 2625. Nickerson, B.; Jorgenson, J. W. /. Chromatogr. 1989,480, 157. Albin, M.; Weinberger, R; Sapp, E.; Moring, S. Anal. Chem. 1991,63, 417. (12) Butchner, E. C.; Lowry, 0. H. Anal. Biochem. 1976,76,502. (13) Lindroth, P.; Mopper, K Anal. Chem. 1979,51, 1667. (14) For example: KO, W. H.; Suminto, J. T. In Senson; Gopel, W., Hasse, J., Zemmel, J. N., Eds.; VCH: Weinhein, Germany, 1989 Vol. 1,pp 107-168.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994 4127
analyte reservoir
reagent reservoir
reservoir chamber
i
cross
+ 5 mm
waste reservoir
cover slip
substrate
Figure 1. Schematic of the microchip with integrated precolumn reactor. The reaction chamber is 2 mm long, and the separation column is 15.4 mm long.
photomask was fabricated by sputtering chrome (50 nm) onto a glass slide and ablating the microchip design (Figure 1) into the chrome film using a CAD/CAM excimer laser machining system (ArF, 193 nm; Resonetics, Inc.). The column design was then transferred onto the substrates using a positive photoresist (Shipley 1811). The channels were etched into the substrate in a dilute, stirred HF/NH4F bath. To form the closed network of channels, a cover plate was bonded to the substrate over the etched channels by use of a direct bonding te~hnique.~ Cylindrical glass reservoirs were affixed on the substrate using epoxy. Platinum electrodes provided electrical contact from the power supply (Spellman CZE1000R) to the solutions in the reservoirs. The reaction chamber was designed to be wider than the separation column to give lower electric field strengths in the reaction chamber and thus longer residence times for the reagents. Figure 2a shows a scanning electron microscope (SEM) image of the injection cross and the lower portion of the reaction chamber prior to bonding of the cover plate. The image has the same orientation as the microchip schematic in Figure 1. The reaction chamber is 96 pm wide at half-depth and 6.2 pm deep, and the separation column is 31 pm wide at half-depth and 6.2 pm deep. Figure 2b shows an SEM image of the cross section of the separation channel. The microchip was broken perpendicular to the separation column %lmm downstream from the injection cross. The isotropic etch of the glass substrate, i.e., uniform etch in all directions, is evident in the trapezoidal geometry of the cross section. Column performance and separations were monitored onmicrochip using a singlepoint detection scheme via laser-induced fluorescence (LIF). An argon ion laser (351.1 nm, 10 m W Coherent Innova 90) was used for excitation and focused to a spot onto the microchip using a lens (100 mm focal length). The fluorescence signal was collected using a 20x objective lens, followed by spatial filtering (0.6 mm diameter pinhole) and spectral filtering (440 nm bandpass, 10 nm bandwidth), and measured using a photomultiplier tube (PMT; Oriel 77340). The schematic 4128 Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
I
I
Figure 2. (a, top) Scanning electron microscope (SEM) image of injection cross. The broader channel is the lower portion of the reaction chamber. The separation channel is 6.2 pm deep and 31 pm wide, and the reaction chamber is 6.2 pm deep and 96 pm wide. The SEM is in the same orientation as Figure 1. (b, bottom) SEM image of cross section of separation column after bonding of coverplate to the substrate.
in Figure 3a shows the data acquisition/voltage switching a p paratus, which is computer controlled using programs written inhouse in Labview 3.0 (National Instruments). The compounds used for the experiments were arginine (0.48 mM) , glycine (0.58 mM), and o-phthaldialdehyde (5.1 mM; Sigma Chemical Co.). The buffer in all of the reservoirs was 20 mM sodium tetraborate with 2% (v/v) methanol and 0.5% (v/v) 2-mercaptoethanol. 2-Mercap toethanol is added to the buffer as a reducing agent for the derivatization reaction. lo Figure 3b shows the ratio of the potentials at each of the reservoirs for a given potential applied to the system. This configuration allowed the lowest potential drop across the reaction chamber (25 V/cm for 1.0 kV applied to the microchip) and the highest across the separation column (300 V/cm for 1.0 kV applied to the microchip) without significant bleeding of the product into the separation column. The voltage divider used to establish the potentials applied to each of the reservoirs had a total resistance of 100 with 10 MQ divisions. The sample and reagent are electroosmotically pumped into the reaction chamber with a volumetric ratio of 1:1.06. Therefore, the solutions from the analyte and reagent reservoirs are diluted by a factor of ~ 2 Buffer .
microchip
laser
analyte ,/ r e a p t ('waste I
-.
f
-
IwOaste 'buffer
i
switch
PCWI Labview
trigger
--
-acquisition
5 1PI Analyte
Buffer +HV
6kl current amplifier
Reagent
-
--c
Analyte Waste +0.2Hv
+I
time
2o
Figure 3. (a, top) Diagram of the high-voltage switching apparatus and detectioddata acquisition system. (b, bottom) Block diagram of flow pattern for precolumn reactor microchip with the relative potentials applied at each reservoir (arrows depict direction of flow).
RESULTS AND DISCUSSION Fluid manipulation, including sample injections, is performed by simply controlling the potentials applied to the reservoirs; Le., no valves or pumps are required. This allows reagents to be mixed accurately and samples to be injected reproducibly onto the separation column. Using the gated injection scheme described above, Figure 4 shows four injections of glycine derivatized with OPA for increasing injection times of 0.2, 0.4, 0.6, and 0.8 s with an injection field strength of 0.6 kV/cm. The injection field strength is the electric field strength in the separation column during the injection; Le., the high-voltage switch in Figure 3a is open. Similar profiles were obtained for arginine. The injections were observed in the separation column 0.05 mm downstream from the injection cross using the single-point detection scheme. The peak fronts coincide as expected, and the durations of the injection profiles correspond to the injection times. (15) Jorgenson, J. W.; Lukacs, IC D. Anal. Chem. 1981,53,1298.
1.5
1.0
[SI
Figure 4. Series of injection profiles for injection times of (a) 0.2, (b) 0.4, (c) 0.6, and (d) 0.8 s with 6 n j = 0.6 kV/cm and Lobs = 0.05 mm.
I
was simultaneously pumped by electroosmosis from the buffer reservoir toward the waste and analyte waste reservoirs. This buffer stream prevents the newly formed product from bleeding into the separation column. A gated injection scheme, described previously? is used to inject effluent from the reaction chamber onto the separation column. The potential at the buffer reservoir is simply floated (opening of the high-voltage switch in Figure 3a) for a brief period of time (0.1-1.0 s), and sample migrates into the separation column as in an electrokinetic inje~ti0n.l~ To break off the injection plug, the potential at the buffer reservoir is reapplied (closing of the high-voltage'switch in Figure 3a). The length of the injection plug is a function of both the time of the injection and the electric field strength. With this configuration of applied potentials, the reaction of the amino acids with the OPA continuously generates fresh product to be analyzed.
0.5
0.0
i
&?
Fi
l5o /
1
t .
0.0
0.2
0.4
0.6
injection time
0.8
1.0
[SI
Figure 5. Reproducibility of injections as percent relative standard deviation (% rsd) of the peak area with injection time for arginine (e) and glycine ( 0 )at .&j = 1.2 kV/cm and for arginine (A)and glycine (m) at &j = 0.6 kV/cm with Lobs = 0.05 mm. Dashed line represents 2% rsd.
A signiticant shortcoming of many capillary electrophoresis experiments has been the poor reproducibility of the injections. Here, because the microchip injection process is computer controlled, and the injection process involves the opening of a single high-voltage switch, the injections can be accurately timed events. Figure 5 shows the reproducibility of the amount injected (percent relative standard deviation, % rsd, for the integrated areas of the peaks) for both arginine and glycine at injection field strengths of 0.6 and 1.2 kV/cm and injection times ranging from 0.1 to 1.0 s. For injection times of 20.3 s, the percent relative standard deviation is below 1.8%.This is comparable to reported values for commercial, automated capillary electrophoresis instruments.16 However, injections made on the microchip are =lo0 times smaller in volume, e.g., 100 pL on the microchip versus 10 nL on a commercial instrument. Part of this fluctuation is due to the stability of the laser, which is ~ 0 . 6 % For . injection times of (16) For example, Hewlett-Packard reports 1-2% rsd for peak area measure ments.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
4129
3.0
2.0
1.0 0
2
4
time
0.0 0.0
0.2
0.4
0.6
injection time
0.8
1.0
8
Figure 7. Overlay of three electropherograms of arginine and glycine using precolumn derivatization with ephthaldialdehyde with Esep = 1.8 kV/cm and Lsep= 10 mm.
[SI
Figure 6. Variation of injection widths with injection time for arginine (+) and glycine (0)at Enj= 1.2 kV/cm and for arginine (A)and glycine (W) at El,,, = 0.6 kV/cm with Lobs = 0.05 mm. Lines represent best fit for injection times of r 0 . 2 s for arginine and 20.3s for glycine.
20.3 s, the error appears to be independent of the compound injected and the injection field strength. In Figure 6, the injection plug lengths at half-height are plotted versus the injection time. For injection times of 20.2 s for arginine and 20.3 s for glycine at the two selected injection field strengths, the lengths of the injected plugs are a linear function of the injection times and injection field strengths. Because the injection is electrophoretically biased, and arginine has a higher electrophoretic mobility than glycine, injection widths are linear at shorter injection times for arginine. The injection widths are not proportional to the injection times for shorter injection times. The injection cross region has a finite width, and once filled with analyte, the area must be swept clean after the injection. Lines with a y-intercept equal to zero are fitted to the data for injection times of 20.2 s for arginine and 20.3 s for glycine. The slopes of the lines are 3.13 and 1.51 mm/s for arginine with injection field strengths of 1.2 and 0.6 kV/cm, respectively, and 1.44 and 0.692 mm/s for glycine with injection field strengths of 1.2 and 0.6 kV/ cm, respectively. For both arginine and glycine, the slopes for the injection field strength of 1.2 kV/cm are 2 times greater than at 0.6 kV/cm. For the experiments described below, the injector was operated in the reproducible (Figure 5) and linear ranges (Figure 6), and the injection times were scaled to give comparable peak widths and, thus, injection volumes for separations performed at different separation field strengths. The electrophoretic bias in the injections requires the injection time to be long enough to inject accurately the compound with the lowest electrophoretic mobility, e.g., glycine. This, in turn, means compounds with a higher electrophoretic mobility, e.g., arginine, are injected in relative excess. Figure 7 shows the overlay of three electrophoretic separations of arginine and glycine after on-microchip precolumn derivatization with OPA with a separation field strength of 1.8 kV/cm and a separation length of 10 mm. The separation field strength is the electric field strength in the separation column during the separation; Le., the high voltage switch in Figure 3a is closed. The field strength in the reactor is 150 V/cm. The reaction times 4130
6
[SI
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
for the analytes are inversely related to their mobilities; e.g., for arginine the reaction time is 4.1 s and for glycine the reaction time is 8.9 s. The volumes of the injected plugs were 150 and 71 pL for arginine and glycine, respectively, which correspond to 35 and 20 fmol of the amino acids injected onto the separation column. The gated injector allows rapid sequential injections to be made. In this particular case, an analysis could be performed every 4 s. The observed electrophoretic mobilities for the compounds are determined by a linear fit to the variation of the linear velocity with the separation field strength. The slopes were 29.1 and 13.3 mm2/(kV-s) for arginine and glycine, respectively. No evidence of Joule heating was observed, as indicated by the linearity of the velocity versus field strength data. A linear fit produced correlation coefficients of 0.999 for arginine and 0.996 for glycine for separation field strengths from 0.2 to 2.0 kV/cm. With increasing potentials applied to the microchip, the field strengths in the reaction chamber and separation column increase. This leads to shorter residence times of the reactants in the reaction chamber and faster analysis times for the products. By varying the potentials applied to the microchip, the reaction kinetics can be studied. The variation in amount of product generated with reaction time is plotted in Figure 8. The response is the integrated area of the peak corrected for the residence time in the detector observation window and photobleaching of the product (discussed below). The offset between the data for the arginine and the glycine in Figure 8 is due primarily to the difference in the amounts injected, Le., different electrophoretic mobilities, for the amino acids. A 10-fold excess of OPA was used to obtain pseudo-first-order reaction conditions. The slopes of the lines fitted to the data correspond to the rates of the derivatization reaction. The slopes are 0.13 s-l for arginine and 0.11 for glycine, corresponding to half-times of reaction of 5.1 and 6.2 s, respectively. These half-times of reaction are comparable to the 4 s previously reported for alanine.12 We have found no previously reported data for arginine or glycine. The reaction time varies with the potential applied to the microchip. For lower field strengths in the reaction chamber, the reaction time is longer and should yield more product and larger signals. In Figure 9, the mass detection limits (signal-to-noise ratio (S/N) equal to 2) are estimated for a series of reaction times for arginine and glycine. For longer reaction times, the mass detection limits improve as expected. Detection limits of 0.55 fmol
0.0
-
-1.0
a
v)
8
P 8
& -2.0
F
-3.0
10
0
20
r e a c t i o n time
30
[SI
Figure 8. Variation of product formation (In [response])with reaction time for arginine (0)and glycine (m)derivatized with ephthaldialdehyde. Lines represent linear fits. Error bars are f o for three runs.
'I l
!i ii .
0.5
4'
T
1
Hdet f
Hdiff
(2)
(3)
I
(4)
t 0
Htod= Hhj f
where Hinj,Hdet,and Hdiffare the contributions to the plate height from the injection plug length, the detector observation length, and axial diffusion, respectively. The contributions from the injection plug length and the detector observation length are time independent, and the contribution to the plate height from axial diffusion is time dependent. Effects such as Joule heating are not considered because the variation of the linear velocity with the separation field strength is linear, as mentioned above. The contributions to the plate height from the injection plug length and the detector observation length are17
1.5 r
where k is a geometrical factor, Zis the intensity of the laser ( ~ 4 0 0 W/cm2>, UF is the fluorescence cross section, N is the number of molecules in the detection volume, At is the detector residence time (0.01-0.1 s for arginine and 0.02-0.25 s for glycine), and t is the photochemical lifetime. k, I, UF, and N are constant for the experiments. The residence time of the product in the detection volume is known, and thus, the photochemical lifetime, z, of the reaction product can be estimated by use of a linear fit of the variation of In S with At. The photochemical lifetimes are 51 ms for arginine and 58 ms for glycine under these experimental conditions. The products are known to have poor stability,I3and not surprisingly,the photochemical lifetimes are short compared to the longer observation times. Accurate quantitative measurements must take account of the photobleaching effect. To improve the peak capacity of the separation, the band dispersion needs to be m i n i i e d . For electrophoretic separations the total plate height can be expressed as
,
20
,
,
,
,
40
60
reaction t i m e
[SI
,
,
.
/
.
80
Figure 9. Variation of detection limits (SIN = 2) with reaction time for arginine (0)and glycine (m) derivatized with o-phthaldialdehyde. Error bars are fa for three runs.
for arginine and 0.83 fmol for glycine are reached for reaction times of 36 and 80 s, respectively. The difference in detection limits between arginine and glycine is assumed to be differences in relative fluorescence and in separation efficiency. The source of the limiting background for this experiment is fluorescence from the glass substrate. Detection limits could be improved by using fused silica substrates, which would minimize the background fluorescence. Photobleaching can affect quantitative results in fluorescence measurements. The degree of photobleaching was estimated by determining the photochemical lifetimes of the fluorescent products. The fluorescence signal was measured as a function of the residence time in the detector observation window by running a frontal electropherogram of the individual products. The fluorescence signal, S, for the continuous stream measurements is
where lhj and &t are the lengths of the injection plug and detector observation, respectively, and LP is the separation length. The contributions from the injection plug length (see injection profiles in Figure 4) and detector observation length are written as Gaussian functions. The lengths of the injection plug and the detector observation are constant for all experiments. If these time-independent contributions predominate in their contribution to the plate height, then the total plate height decreases as the separation length increases. The contribution to the plate height from axial d f i s i o n is1*
Hdiff= 2Dm/(ME)
(5)
where p is the effective electrophoretic mobility of the analyte, E is the electric field strength, and Dmis the diffusion coefficient of the analyte in the buffer. The contribution from axial diffusion to the plate height is reduced by increasing the separation field strength and, consequently, reducing the analysis time. (17) Stemberg, J. C. Adv. Chromatogr. 1966, 2, 205. (18) Giddings, J. C. Dynamics ofChromatography,Part I: Principles and Theoy; Marcel Dekker: New York, 1965; Chapter 2.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
4131
especially at the channel walls. As seen in the SEM image in Figure 2a, the separation channel walls have a noticeable roughness. These etch defects derive primarily from the imperfections in the photomask. The photomask that was generated using W ablation of a thin chrome film had an edge smoothness of 6 1 pm on a 25 pm wide line. This roughness in the mask is then transferred to the photoresist during exposure and, subsequently, etched into the substrate. In Figure 10, the plate height increases with increasing electric field strength. If related to the surface roughness, the magnitude of such a contribution is difficult to quantitate since the exact nature of the channel surface is not known. Prior work at comparable separation field strengths yielded more favorable results and agrees with eq 2.6 However, in that work, an e-beam written photomask, which had edge smoothness more than 1 order of magnitude better, was used for the microchip fabrication. The threshold of roughness that can be tolerated for the photomask and thus the channel wall is under further investigation.
v al
a
w
a
0 0.0
0.5
1.0
1.5
2.0
separation field strength [kV/cml Figure 10. Variation of plate height with the separation field strength for arginine (0)and glycine (M) derivatized with ephthaldialdehyde with Lsep= 10 mm. The contribution to the plate height from the injection and detection is calculated from eqs 3 and 4 for arginine (A)and glycine (+). Error bars are f a for three runs.
In Figure 10, the total plate height versus the electric field strength is plotted along with the calculated contributions from the injection plug length and the detector observation length. The time-independent contributions differ for arginine and glycine because the injections have an electrophoretic bias, and consequently, compounds with a greater electrophoretic mobility will have a larger contribution to the plate height. The injection plug lengths at half-height were 0.80 and 0.37 mm for arginine and glycine, respectively, which correspond to Hi,,equal , to 11.6 and 2.5 pm. These contributions are substantial, but in order to run the injector in the linear, reproducible regime for all separation field strengths, longer injection times were required. The detector observation length, Le., the laser spot size, was ~ 5 pm, 0 which corresponds to &et equal to 21 nm. In the range of separation field strengths used, the contribution from axial dispersion is small relative to the other contributions to the total plate height and is not discussed further. Unfortunately, as the field strength increases, the total plate height increases. This behavior is reminiscent of liquid chromatography, where mass transfer in the mobile and/or stationary phases dominates at high mobile phase velocities. The contribution from mass transfer to the plate height is a linear function of the mobile phase velocity:
H,, = Cu
(6)
where u =pE (7) u is the linear velocity of the mobile phase and C is the coefficient
of mass transfer. Typically, in capillary electrophoresis, contributions to the plate height from mass transfer are neglected because there should be no interaction of the analyte with the walls of the capillary. However, in Figure 10 the magnitude of this contribution is surprisingly large especially for small molecules which tend to have little or no interaction with the walls. An alternative explanation might be that the source of band broadening is due to inhomogeneity of the channel surface, 4132 Analytical Chemistry, Vol. 66, No. 23, December 7, 7994
CONCLUSION Chemical reactions followed by analysis of the products have been demonstrated on a single microchip. Devices can be tailored to accommodate any such reaction to be performed in a miniaturized environment. The reactor volumes and separation lengths can be optimized accordingly. Electroosmotically driven flow enables buffer, analyte, and reagent streams to be controlled precisely in the channel manifold of the microchip without the use of valves or pumps. The gated injector allows continuous loading of sample and rapid sequential injections to be made. Law dead volume connections between channels can be easily fabricated allowing pre- and postseparation reactions to be incorporated on-microchip. Having an electrode placed at the injection cross would allow independent control of the potentials in the reaction chamber and separation column. To have a long reaction time for the chemical reaction, the field strength in the reaction chamber should be minimized, but for capillary electrophoresis, the resolution between compounds increases with increasing electric field strength. With the current design, a compromise must be reached with the potentials applied to the microchip so that the OPA has sufficient time to react with the amino acids without seriously impeding the quality of the separation. ACKNOWLEDGMENT This research was 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 DE-AC05840R.21400. Also, this research was sponsored in part by an appointment for S.C.J. to the Alexander Hollaender Distinguished Postdoctoral Fellowship Program sponsored by the U.S. DOE and for A.W.M. and R.H. 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 August 1, 1994. Accepted September 26, 1994.@ 'Abstract published in Advance ACS Abstracts, October 15. 1994
