Anal. Chem. 1994, 66. 3472-3476
`
`Microchip Capillary Electrophoresis with an Integrated
`Postcolumn Reactor
`Stephen C. Jacobson, Lance B. Koutny, Roland Hergenroder, Alvin W. Moore, Jr., and J. Michael Ramsey'
`Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008,
`Oak Ridge, Tennessee 37831-6142
`
`A glass microchip with a postcolumn reactor was fabricated
`to conduct postseparation derivatization using o-phthaldial-
`dehyde as a fluorescent “tag” for amino acids. This miniatur-
`ized separation device was constructed using standard pho-
`tolithographic, wet chemical etching, and bonding techniques.
`Effects of the reagent stream on separation efficiency were
`In addition, a novel gated injector was demon-
`investigated.
`strated which maintains the integrity of the analyte, buffer,
`and reagent streams.
`
`For capillary separation systems, the small band volumes
`can limit the number of viable detection schemes. Fluorescence
`detection remains one of the most sensitive detection techniques
`for capillary electrophoresis.1 11When incorporating direct
`fluorescence detection into a system that does not have
`naturally fluorescing analytes, derivatization of the analyte
`must occur either pre- or postseparation. When the fluorescent
`“tag” is short lived or the separation is hindered by presepa-
`ration derivatization, postcolumn addition of derivatizing
`reagent becomes the method of choice. A variety of post-
`column reactors have been demonstrated for capillary
`electrophoresis.2-7 However, the ability to construct a post-
`column reactor with extremely low volume connections to
`minimize band distortion has been difficult. We have taken
`the approach of fabricating a microchip device for electro-
`phoretic separations with postcolumn reactions using standard
`microchip fabrication techniques. The injector, separation
`column, and reaction column can be coupled in a single
`monolithic device, enabling extremely low volume exchanges
`between individual column functions. This microfabrication
`approach is a part of the continuing effort toward microma-
`chining of miniaturized instrumentation for chemical separa-
`tions which includes devices for gas chromatography,8 liquid
`chromatography,9-10 and capillary electrophoresis.11-16
`
`(1) For example, see: Ruhr, W. G.; Monnig, C. A. Anal. Chem. 1992,64, 389R.
`(2) Pentoney, S.; Huang, X.; Burgi, D.; Zare, R. Anal. Chem. 1988, 60, 2625.
`(3) Tsuda, T.; Kobayashi, Y.; Hori, A.; Matsumoto, T.; Suzuki, O. J. Chromatogr.
`1988, 456, 375.
`(4) Rose, D. J., Jr.; Jorgenson, J. W. J. Chromatogr, 1988, 447, 117.
`(5) Nickerson, B.; Jorgenson, J. J. Chromatogr. 1989, 480, 157.
`(6) Rose, D. J., Jr. J. Chromatogr. 1991, 540, 343.
`(7) Albin, M.; Weinberger, R.; Sapp, E.; Moring, S. Anal. Chem. 1991, 63, 417.
`(8) Terry, S. C.; Jerman, J. H.; Angell, J. B. IEEE Trans. Electron Devices 1979,
`26, 1880.
`(9) Manz, A.; Miyahara, Y.; Miura, J.; Watanabe, Y.; Miyagi, H.; Sato, K. Sens.
`Actuators 1990, Bl, 249.
`(10) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem.
`1994, 66, 2369.
`(11) Manz, A.; Harrison, J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Ludi,
`H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253.
`(12) Harrison, D. J.; Manz, A.; Fan, Z.; Ltidi, H.; Widmer, H. M. Anal. Chem.
`1992, 64, 1926.
`(13) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993, 65, 1481.
`3472 Analytical Chemistry; Vol. 66, No. 20, October 15, 1994
`
`In this paper, we describe a first generation postcolumn
`reactor on a microchip. Broadening of the analyte band due
`to conventional mechanisms including injection plug length,
`detector observation length, and axial diffusion and the added
`influence of combining of the separation effluent and reagent
`stream of the fluorescent tag, o-phthaldialdehyde (OPA),17
`In addition, a new approach to analyte injection
`are studied.
`was developed to isolate the analyte, running buffer, and
`reagent streams. Previous flow designs used for electrophoretic
`sample loading and separation on microchip devices for
`capillary electrophoresis1 ’’14’15 are difficult to implement with
`a postcolumn reactor. The operating conditions and benefits
`of the new injection scheme are discussed.
`
`EXPERIMENTAL SECTION
`The microchip was fabricated using standard photolitho-
`graphic, wet chemical etching, and bonding techniques. A
`photomask was fabricated by sputtering chrome (50 nm) onto
`a glass slide and ablating the column design (Figure 1) into
`the chrome film via a CAD/CAM laser machining system
`(Resonetics, Inc., Nashua, NH). The column design was then
`transferred onto the substrates using a positive photoresist
`(Shipley 1811, Newton, MA). The channels were etched into
`the substrate in a stirred, dilute HF/NH4F bath for 20 min.
`To form the separation column, a coverplate was bonded to
`the etched channels using a direct bonding
`the substrate over
`technique.15 The surfaces were hydrolyzed in dilute NH4-
`OH/H2O2 solution, rinsed in deionized, filtered H2O, joined,
`and then annealed at 500 °C. Cylindrical glass reservoirs
`were affixed on the substrate using RTV silicone (General
`Electric, Waterford, NY). Platinum electrodes provided
`from the power supply (Spellman
`electrical contact
`CZE1000R, Plainview, NY) to the solutions in the reservoirs.
`The dimensions of the columns on the microchip are labeled
`in Figure 1. Because the substrate is glass and the channels
`are chemically wet etched, an isotropic etch occurs, i.e., the
`glass etches uniformly in all directions, and the resulting
`channel geometry is trapezoidal. The channel cross section
`has dimensions of 5.2 jtm deep, 57 /zm wide at the top, and
`45 jtm wide at the bottom. The dimensions were obtained
`using a profilometer (Alpha-Step 200, Tencor Instruments,
`Mountain View, CA) after etching of the substrate and prior
`
`(14) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A.
`Science 1993, 261, 895.
`(15) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Warmack, R. J.; Ramsey,
`J. M. Anal. Chem. 1994, <55, 1107.
`(16) Jacobson, S. C.; Hergenrfider, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem.
`1994, 66, 1114.
`(17) Roth, M. Anal. Chem. 1971, 43, 880.
`
`0003-2700/94/0366-3472$04.50/0
`&copy; 1994 American Chemical Society
`
`Downloaded via UNIV OF VIRGINIA on November 6, 2018 at 17:54:12 (UTC).
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`Figure 1. Schematic of the microchip with postcolumn reactor.
`
`to bonding of the coverplate. The profilometer uses a stylus
`to provide a one-dimensional depth profile of the substrate
`surface.
`Column performance and separations were monitored on-
`microchip via fluorescence using an argon ion laser (351.1 nm
`for amino acid/OPA, 10 mW, and 514.5 nm for rhodamine
`B, 50 mW; Coherent Innova 90, Palo Alto, CA) for excitation.
`The fluoresence signal was collected with a photomultiplier
`tube (PMT; Oriel 77340, Stratford, CT) for point detection
`and a charge coupled device (CCD; Princeton Instruments,
`Inc. TE/CCD-512TKM, Trenton, NJ) for imaging a region
`of the microchip.15 The compounds used for the experiments
`were rhodamine B (Exciton Chemical Co., Inc., Dayton, OH),
`arginine, glycine, threonine, and o-phthaldialdehyde (Sigma
`Chemical Co., St. Louis, MO). A sodium tetraborate buffer
`(20 mM, pH 9.2) with 2% (v/v) methanol and 0.5% (v/v)
`/3-mercaptoethanol was the buffer in all experiments. The
`concentrations of the amino acid, OPA, and rhodamine B
`solutions were 2 mM, 3.7 mM, and 50/iM, respectively. Several
`run conditions were utilized for microchip diagnostics and
`will be described as needed.
`
`RESULTS AND DISCUSSION
`Because several separation lengths were used to study
`different aspects of the microchip performance, the efficiencies
`will be reported primarily using the plate height (H). The
`contributions to the plate height are18'19
`H = Hm + Hm + Hia = 2^
`
`2
`
`/
`det
`12L.„
`
`(1)
`K>
`
` nj
`12L,
`
`  +
`
`u
`
`Figure 2. Variation of plate height (H) with the separation field strength
`= 6 (•) and 8 mm ( ). Error bars are
`(E„p) for rhodamine B with
`±cr.
`
`of the analyte in the buffer, u is the linear velocity of the
`is the detector
`is the injection plug length, /det
`analyte, /i„j
`observation length, and Liip is the separation length. The
`effects of Joule heating were not considered because the power
`dissipation was below 1 W/m for all experiments.20 The
`contribution from the axial diffusion is time dependent, and
`the contributions from the injection plug length and detector
`In electrophoretic
`observation length are time independent.
`separations, the linear velocity of the analyte, u, is equal to
`the product of the effective electrophoretic mobility, ntp, and
`the electric field strength, E.
`To test the band broadening effects of reagent addition at
`the mixing tee, a fluorescent laser dye, rhodamine B, was used
`as a probe. Efficiency measurements calculated from peak
`widths at half-heights were made using the point detection
`scheme at distances of 6 and 8 mm from the injection cross,
`1 mm upstream and 1 mm downstream from the mixing
`or
`tee. The results are plotted in Figure 2 as the plate height
`the electric field strength in the separation column
`versus
`(/ssep). Rhodamine B was injected onto the column using the
`pinched sample loading method15 to ensure that the contribu-
`tion of the injection plug length to the plate height remained
`the measurements (//jnj = 0.23 and 0.17 fim for
`constant over
`the separation lengths of 6 and 8 mm, respectively). Single
`point detection was used, and the column length observed by
`the detector is equal to the laser spot size, =60 ftm (//dct =
`0.05 and 0.04 ^m for the separation lengths of 6 and 8 mm,
`respectively). The time-independent contributions are small
`relative to the total plate height for these experiments.
`The electric field strengths in the reagent column and the
`separation column were approximately equal, and the field
`strength in the reaction column was a factor of 2 higher. This
`voltage configuration provided approximately a 1:1 volume
`ratio of reagent stream to effluent from the separation column.
`As the field strengths increase, the degree of turbulence at the
`
`the contributions of axial
`where //diff, H\ns, and //det are
`diffusion, injection plug length, and detector observation length
`to the plate height, respectively. Dm is the diffusion coefficient
`
`(18) Giddings, J. C. Dynamics of Chromatography, Parti: Principles and Theory,
`Marcel Dekker: New York, 1965; Chapter 2.
`(19) Sternberg, J. C. Adv. Chromatogr. 1966, 2, 205.
`(20) Monnig, C. A.; Jorgenson, J. W. Anal. Chem. 1991, 63, 802.
`
`Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
`
`3473
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`positive slope from the mixing tee to the waste reservoir because
`the amino acids continue to react as they travel down the
`In the observation window, the residence time of the
`column.
`product is 8 times longer at the field strength of 240 V/cm
`than at 1920 V/cm. Only a 5-mm region of the reaction
`even illumination of the
`column was observed to ensure
`microchip by the expanded laser beam and efficient collection
`of the fluorescence signal by the CCD.
`Ideally, the observed
`fluorescence from the product would have a step function for
`a response following the mixing of the separation effluent and
`derivatizing reagent. However, the kinetics of the reaction
`and a finite rate of mixing dominated by diffusion prevent
`this from occurring.
`A different injection scheme was developed for the post-
`column reactor microchip in order to keep the analyte, buffer,
`and reagent streams isolated. For the postcolumn reaction
`separations, the microchip was operated in a continuous sample
`loading/separation mode whereby the sample was continuously
`pumped from the analyte reservoir through the injection cross
`toward the analyte waste reservoir. Buffer was simultaneously
`pumped from the buffer reservoir toward the analyte waste
`and waste reservoirs to deflect the analyte stream and prevent
`the sample from migrating down the separation column. To
`inject a small aliquot of sample, the potentials at the buffer
`and analyte waste reservoir are simply floated for a short
`period of time (=100 ms), allowing sample to migrate down
`the separation column as in an electrokinetic injection.22 To
`break off the injection plug, the potentials at the buffer and
`analyte waste reservoir are reapplied. A shortfall of this
`method is that the composition of the injected plug has an
`electrokinetic bias whereby the faster migrating compounds
`are introduced preferentially into the separation column over
`slower migrating compounds. The schematic in Figure 4a
`shows the electric potential distributions used when 1 kV is
`applied to the entire system. With this voltage configuration,
`the electric field strengths in the separation column (Esep) and
`the reaction column (Erxn) are 200 and 425 V/cm, respectively.
`This allows the combining of 1 part separation effluent with
`1.125 parts reagent at the mixing tee. A sample introduction
`system such as this, with or without postcolumn reaction, allows
`a very rapid cycle time for multiple analyses.
`In Figure 4b-d, the gated injection at the injection cross
`is imaged by the CCD camera with the potential configuration
`in Figure 4a. The analyte being pumped through the microchip
`was rhodamine B (shaded area), and the orientation of the
`CCD images of the injection cross is the same as in Figures
`1 and 4a. Figure 4b shows the analyte being pumped through
`the injection cross prior to the injection. Between Figures 4b
`and 4c, the potentials at the buffer and analyte waste reservoirs
`are floated for 100 ms. At this point, the analytes migrate
`down the separation column according to their electrophoretic
`mobilities. The potentials at the buffer and analyte waste
`reservoirs are reapplied prior to the CCD image taken in Figure
`4c. Figure 4c catches the analyte plug being broken away
`from the analyte stream and being injected into the separation
`column. Figure 4d shows the analyte plug moving away from
`the injection cross after an injection plug has been completely
`introduced into the separation column. By Figure 4d, the
`
`distance (mm)
`Figure 3. Variation of laser-induced fluorescence (LIF) Intensity with
`distance downstream from the mixing tee for glycine derivatlzed with
`o-phthaldialdehyde with Enn = 240, 480, 960, and 1920 V/cm.
`
`mixing tee appears to increase. At the separation distance of
`6 mm (1 mm upstream from the mixing tee), the plate height
`data decrease as expected, i.e., with the inverse of the linear
`velocity of the analyte (eq 1). At the 8-mm distance (1 mm
`downstream from the mixing tee), the plate height decreases
`as the field strength increases from 140 to 280 V/cm as
`expected but increases in the 280-840 V/cm range. This
`behavior is abnormal (eq 1) for typical capillary electrophoresis
`experiments and demonstrates a band broadening phenomenon
`turbulence for converging electroosmotically pumped
`or
`streams. The geometry of the mixing tee was not optimized
`to minimize this band distortion. Above the separation field
`strength of 840 V/cm, the system stabilizes, and again the
`plate height decreases with increasing linear velocity. For
`Esep = 1400 V/cm, the plate height at the 8-mm distance is
`60% greater than that at 6 mm. Efficiency losses at typical
`field strengths used in capillary electrophoresis (<250 V/cm)
`appear to be minimal.
`Following the combining of the two streams at the mixing
`tee, the intensity of the fluorescence signal generated from
`the reaction of the OPA with an amino acid was tested by
`continuously pumping glycine down the column as a frontal
`electropherogram to mix with the OPA at the mixing tee. The
`fluorescence signal from the OPA/amino acid reaction was
`collected using a CCD camera
`as the reaction occurred
`downstream from the mixing tee (Figure 3). Again, the
`relative volume ratio of the OPA and glycine streams was
`=1:1. OPA has a typical half-time of reaction with amino
`acids of 4 s.21
`In Figure 3, the average residence times of an
`analyte molecule in the window of observation (0-4 mm) are
`4.68, 2.34, 1.17, and 0.58 s for the electric field strengths in
`the reaction column (Erxn) of 240,480,960, and 1920 V/cm,
`respectively. The relative intensities of the fluorescence
`correspond qualitatively to this 4-s half-time of reaction. As
`the field strength increases in the reaction column, the slope
`of the intensity of the fluorescence decreases because the
`glycine and OPA are swept away from the mixing tee faster
`with higher field strengths. The fluorescence signal has a
`
`(21) Butchner, E. C.; Lowry, O. H. Anal. Biochem. 1976, 76, 502.
`
`(22) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298.
`
`3474 Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
`
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`

`0
`
`2
`
`4
`time [s]
`Figure 5. Electropherograms of (a) arginine and glycine and (b) arginine
`and threonine using postcolumn derivatization with o-phthaldialdehyde
`with E„p = 800 V/cm and
`= 13.5 mm.
`p
`
`6
`
`Table 1. Retention and Efficiency Data for Electropherograms In
`Figure 5
`H(v m)
`N
`compd
`
`'r (s)
`
`arginine
`glycine
`
`arginine
`threonine
`
`Figure 5a
`
`Figure 5b
`
`2.64
`4.11
`
`2.59
`4.47
`
`1320
`329
`
`1300
`2460
`
`10.2
`41.0
`
`10.4
`5.5
`
`The electropherograms in Figure 5 demonstrate the
`separation of two pairs of amino acids. The voltage con-
`figuration is the same as in Figure 4a, except the total applied
`voltage is 4 kV, which corresponds to an electric field strength
`of 800 V/cm in the separation column (Exp) and 1700 V/cm
`in the reaction column (Ertn). The injection times were 100
`ms for the experiments which correspond to estimated injection
`plug lengths of 384, 245, and 225 nm (Hinj = 0.91,0.37, and
`0.31 /am) for arginine, glycine, and threonine, respectively.
`The injection volumes of 102, 65, and 60 pL correspond to
`200, 130, and 120 fmol injected for arginine, glycine, and
`threonine, respectively. The point of detection is 6.5 mm
`downstream from the mixing tee, which gives a total column
`length of 13.5 mm for the separation and reaction, and //<ie,
`equals 0.02 /am for a 60-/am laser spot size. Again, the time-
`independent contributions to the total plate height are small
`(Table 1). The efficiencies for the separations involving the
`postcolumn reactions are much lower than those for the
`rhodamine B (H = 1.8 /um for EXf = 840 V/cm and Z.sep
`8 mm in Figure 2). The plate heights for arginine, glycine,
`and threonine were 6, 23, and 3 times those of rhodamine B,
`respectively.
`The reaction rates of the amino acids with the OPA are
`moderately fast but not fast enough on the time scale of these
`experiments. An increase in the band distortion is observed,
`presumably because the mobilities of the derivatized com-
`pounds are different from those of the pure amino acids. Until
`the reaction is complete, the zones of unreacted and reacted
`amino acid will move
`at different velocities, causing a
`broadening of the analyte zone. As evidenced in Figure 5a,
`glycine shows significant broadening with the postcolumn
`
`=
`
`Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
`
`3475
`
`bed
`
`h—I
`60 (im
`(Top, a) Schematic of flow pattern for gated injector (arrows
`Figure 4.
`depict direction of flow).
`(Bottom) CCD image of gated injector using
`rhodamine B (b) prior to injection, (c) during injection of analyte plug,
`and (d) after injection into separation column with
`= 200 V/cm.
`
`loading/separation mode has resumed, and a clean injection
`plug with a length of 142 jim has been introduced into the
`separation column. As seen below,
`the gated injector
`contributes only a minor fraction to the total plate height.
`The injection plug length is a function of the time of the
`injection and the electric field strength in the column. The
`shape of the injected plug is skewed slightly because of the
`directionality of the cleaving buffer flow.
`This gated injector differs from the pinched sample loading
`on several counts. With the gated injector, the sample migrates
`electrophoretically down the separation column and is cleaved
`by restoring the flow of buffer from the buffer reservoir. The
`pinched sample loading pumps the sample through the injection
`cross, and the plug which is injected onto the separation column
`is the sample which resides in the injection cross. With the
`the amount
`introduced onto the
`pinched sample loading,
`separation column is time independent and has no electro-
`is both time
`phoretic bias. The gated sample injector
`dependent and electrophoretically biased. Presently,
`the
`pinched sample loading cannot be run in a continuous sampling
`mode, whereas the gated injector can.
`
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`labeling. To ensure that the excessive band broadening was
`not a function of the migration time, threonine was also tested.
`Threonine has a slightly longer migration time than the glycine
`(Figure 5); however, the broadening is not as extensive as that
`for glycine.
`
`CONCLUSION
`The use of micromachined postcolumn reactors can improve
`the power of postseparation reactions as an analytical tool by
`minimizing the volume of the extracolumn plumbing, especially
`between the separation and reagent columns. This microchip
`design (Figure 1) was fabricated with modest lengths for the
`separation (7 mm) and reaction columns (10.8 mm) which
`were more than sufficient for this demonstration. Longer
`separation columns can be manufactured on a similar size
`microchip using a serpentine geometry15 to perform more
`difficult separations. There are
`a number of potential
`improvements to increase the performance of this initial
`demonstration device. Examples include variation of the
`flow rates and the separation/reaction
`reagent/effluent
`channel widths to reduce flow rates in the reaction channel
`
`and modification of column geometry to improve mixing
`In addition, detection sensitivity can be improved
`efficiency.
`by fabrication on fused silica substrates rather than on glass.
`These improvements are presently being explored in our
`laboratory.
`
`ACKNOWLEDGMENT
`This research was sponsored by U.S. Department of Energy
`(DOE), Office of Research and Development. Oak Ridge
`National Laboratory is managed by Martin Marietta Energy
`Inc., for the U.S. Department of Energy under
`Systems,
`contract DEAC05-840R21400. 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 L.B.K., A.W.M., and
`R.H. to the ORNL Postdoctoral Research Associate Program.
`These programs are administered by the Oak Ridge Institute
`for Science and Education and ORNL.
`
`Received for review April 5, 1994. Accepted June 17, 1994.®
`• Abstract published in Advance ACS Abstracts, August 1, 1994.
`
`3476 Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
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