Anal. Chem. 1994,66,1107-1113
`
`Effects of Injection Schemes and Column Geometry on the
`Performance of Microchip Electrophoresis Devices
`Stephen C. Jacobson/ Roland Hergenroder/ Lance B. Koutny/ R. J. Warmack/ and J. Michael Ramsey*’* 1
`Chemical and Analytical Sciences Division and Health Sciences Research Division, Oak Ridge National
`Laboratory, Oak Ridge, Tennessee 37831-6142
`
`A glass microchip column was fabricated for free-solution
`electrophoresis. The channels were wet chemically etched on
`a substrate using standard photolithographic techniques and
`were sealed using a direct bonding technique. Two methods
`of sample introduction are evaluated employing a cross-type
`channel geometry. With the preferred sample loading method,
`the volume of the sample plug can be accurately controlled and
`is time independent, enabling a constant volume to be injected.
`The injection can be controlled electronically and does not
`induce any electrophoretic mobility based bias. Separations
`were performed on a compact chip with a serpentine column
`geometry that has a 165-mm separation channel in an area
`of less than 10 mm X 10 mm. Band-broadening effects in a
`serpentine column pattern were studied.
`
`The microfabrication of analytical instrumentation provides
`an interesting alternative for chemical sensing. A common
`approach to chemical sensor design is to rely on some general
`physical property observable for quantification, such as an
`electrode current or acoustic wave propagation, and a
`chemically selective coating for chemical specificity.1 Such
`approaches have had limited success with complex mixtures
`of molecular species. The design of high-performance chem-
`ically selective coatings is presently a difficult task. Sensor
`concepts based on conventional
`laboratory approaches to
`chemical measurement problems benefit from a large knowl-
`edge base and thus may be more amendable to a priori designs.
`In addition, microfabricated chemical instruments are likely
`to be more versatile, allowing a single device to address differing
`sensing problems. Microinstrumentation could well derive
`benefits similar to microelectronics, i.e., low cost, compact
`size, high speed,2 and parallel analyses. One avenue for the
`miniaturization of chemical instrumentation is to microma-
`chine a monolithic device using standard lithographic, etching,
`and deposition techniques, i.e., a microchip.3
`Chemical separation techniques appear to be a promising
`area for microinstrumentation. Micromachined columns have
`been fabricated for gas chromatography,4 liquid chromatog-
`raphy,5 and capillary electrophoresis6-9 and evaluated in the
`cases of gas chromatography and capillary electrophoresis.
`* Chemical and Analytical Sciences Division.
`• Health Sciences Research Division.
`(1) Murray, R. W.; Dessy, R. E.; Heineman, W. R.; Janata, J.; Seitz, W. R., Eds.
`Chemical Sensors and Microinstrumentation', ACS Symposium Series 403;
`American Chemical Society, Washington, DC, 1989.
`(2) Jacobson, S. C.; Hergenrflder, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem.,
`following paper in this issue.
`(3) Manz, A.; Fettinger, J. C.; Verpoorte, E.; Lfldi, H.; Widmer, H. M.; Harrison,
`D. J. Trends Anal. Chem. 1991, 10, 144.
`(4) Terry, S. C.; Jerman, J. H.; Angell, J. B. IEEE Trans. Electron Devices 1979,
`26, 1880.
`
`0003-2700/94/0366-1107S04.50/0
`© 1994 American Chemical Society
`
`Capillary electrophoresis10-13 as an analytical
`technique
`appears the most promising because of the experimental
`simplicity and the dependence of the resolution on the electric
`field strength, not the length of the column. Three aspects
`of microchip electrophoresis are addressed in this paper:
`substrate/cover slip bonding, sample introduction, and column
`geometry.
`Previously,6-9 in order to join the substrate with the etched
`channels and a cover plate, the two pieces had to be melted
`together. Another avenue is direct bonding, which allows the
`surfaces of the substrate and the cover slip to be bonded below
`their melting points. Although not necessary for glass, higher
`melting point substrates, e.g., quartz, can be bonded much
`more easily with such a technique.
`Two drawbacks with conventional capillary electrophoresis
`is that the sample introduction method of exchanging sample
`and buffer reservoirs is time consuming and lacks precision,
`and with an electrokinetic injection scheme, ions with greater
`mobilities are disproportionately introduced in larger quan-
`tities. The mechanical exchange of capillaries among various
`reservoirs is of course not possible with a monolithic micro-
`machined device and alternative injection schemes must be
`used. The concept of using electroosmotic flow to perform
`valveless injections on a microchip electrophoresis device was
`previously described.6-7 This injector was essentially a three-
`port device that performed electrokinetic injections without
`mechanical exchange of capillaries but still with mobility-
`based injection bias. An alternative approach for injection
`on a microchip electrophoresis device was investigated that
`eliminates mobility-based injection bias. Two different modes
`of injector operation were studied. The preferred method
`provides an injection volume that becomes time independent,
`leading to greater injection precision.
`We have also studied the impact of a serpentine separation
`channel geometry on separation efficiency. Band-broadening
`phenomena must be understood to optimize the trade-offs
`between compact device designs and separation performance.
`A straight channel design would minimize contributions from
`
`(5) Manz, A.; Miyahara, Y.; Miura, J.; Watanabe, Y.; Miyagi, H.; Sato, K. Sens.
`Actuators 1990, Bl, 249.
`(6) Manz. A.; Harrison, J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Lfldi,
`H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253.
`(7) Harrison, D. J.; Manz, A.; Fan, Z.; Lfldi, H.; Widmer, H. M. Anal. Chem.
`1992, 64, 1926.
`(8) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993, 65, 1481.
`(9) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A.
`Science 1993, 261, 895.
`(10) Hjerten, S. Chromatogr. Rev. 1967, 9, 122.
`(11) Virtanen, R. Acta Polytech. Scand., Appl. Phys. Ser. 1974, No. 123, 1.
`(12) Mikkers, F.; Everaerts, F.; Verheggen, T. J. Chromatogr. 1979, 169, 11.
`(13) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298.
`
`Analytical Chemistry, Vol. 66, No. 7, AprU 1, 1994
`
`1107
`
`Downloaded via UNIV OF VIRGINIA on November 6, 2018 at 17:52:58 (UTC).
`
`See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
`
`Agilent Exhibit 1279
`Page 1 of 7
`
`

`

`I—
`
`I
`
`substrate
`
`Figure 1. Schematic of serpentine channel geometry for the microchip
`with the large circle representing the cover slip and the smaller circles
`the reservoirs.
`
`electric field and geometric effects whereas alternative layouts
`such as the serpentine pattern used here will minimize device
`dimensions.
`
`EXPERIMENTAL SECTION
`The column on the microchip was constructed on a 50 mm
`X 25 mm glass microscope slide (Corning, Inc. No. 2947) and
`covered by a 22-mm circular coverslip. The generation of the
`channels involves standard photolithographic procedures
`followed by chemical wet etching.14 The column image was
`transferred onto the slide with a positive photoresist (Shipley
`1811) and an e-beam written chrome mask (Institute of
`Advanced Manufacturing Sciences, Inc.), and the channel
`was chemically wet etched using a HF/NH4F solution. The
`cover slip was then bonded to the slide using a direct bonding
`technique whereby the slide and cover slip surfaces were first
`hydrolyzed in a dilute NH4OH/H2O2 solution and then joined.
`The chip was annealed at 500 °C in order to ensure proper
`adhesion of the cover slip to the slide. Following the bonding,
`cylindrical plastic reservoirs were affixed to the chip using
`epoxy.
`Figure 1 shows a schematic of the column design. The
`channels are designated by the name assigned in Figure 1
`from the terminus up to the intersection of the four channels,
`and each channel has an accompanying reservoir mounted
`above the channel at the edge of the cover slip. The enclosed
`column length is 9.4 mm from the analyte reservoir to the
`injection cross, 9.4 mm from the analyte waste reservoir to
`the injection cross, 6.4 mm from the buffer reservoir to the
`injection cross, and 171 mm from the injection cross to waste
`reservoir. The radius of all of the turns is 0.16 mm. The cross
`section of
`is seen in the scanning electron
`the channel
`
`(14) Ko, W. H.; Suminto, J. T. In 5>/uorr, Gopel, W., Hasse, J., Zennel, J. N. Eds.;
`VCH: Weinhein, Germany, 1989; Vol. 1, pp 107-168.
`
`1108 Analytical Chemistry, Vol. 66, No. 7, April 1, 1994
`
`Figure 2. Scanning electron microscope image of a cross section of
`the channel. The bar at the bottom represents a length of 11.1 jim.
`
`microscope image in Figure 2. The dimensions of the channel
`are 10 #im deep, 90 pm wide at the top, and 70 pm wide at
`the bottom.
`Two experimental apparatuses were used to analyze chip
`dynamics via analyte fluorescence. A charge coupled device
`(CCD) camera monitored designated areas of the chip and
`a photomultiplier tube (PMT) tracked single-point events.
`The CCD (Princeton Instruments, Inc. TE/CCD-512TKM)
`camera was mounted on a stereomicroscope (Nikon SMZ-
`U), and the chip was illuminated using an argon ion laser
`(514.5 nm, Coherent Innova 90) operating at 3 W with the
`beam expanded to ~20 mm.
`The point detection scheme
`employed a helium/neon laser (543 nm, PMS Electro-Optics
`LHGP-0051) with an electrometer (Keithley 617) to monitor
`response of the PMT (Oriel 77340). The power supplies
`(Spellman CZE1000R) for the electrophoresis were operated
`between +0.3 and +4.4 kV relative to ground.
`The chip was operated under a “sample loading” mode and
`In the sample loading mode, two types
`a “separation” mode.
`of sample introduction were investigated. For a “floating”
`sample loading, a potential was applied to the analyte reservoir
`with the analyte waste reservoir grounded and with the buffer
`and the waste reservoirs floating. For a “pinched” sample
`loading, potentials were applied to the analyte, the buffer,
`and the waste reservoirs with the analyte waste reservoir
`In the
`grounded in order to control the injection plug shape.
`the potential was applied to the buffer
`separation mode,
`reservoir with the waste reservoir grounded and with analyte
`and analyte waste reservoirs at approximately half of the
`potential of the buffer reservoir.
`The analytes used for the diagnostic experiments were
`rhodamineBandsulforhodamine 101 (Exciton Chemical Co.,
`Inc.) at 60 /uM for the CCD images and 6 jiM for the point
`detection. A sodium tetraborate buffer (50 mM, pH 9.2) was
`the mobile phase in all experiments. The laser dyes studied
`are easily separated, but for the sake of column diagnostics,
`the information is invaluable.
`
`Agilent Exhibit 1279
`Page 2 of 7
`
`

`

`Figure 4. Variation of amount of analyte in injection area with time
`for a pinched sample loading (circle) and a floating sample loading
`(square) using rhodamine B.
`
`this flow pattern. Figure 3c shows a floating sample loading.
`The analyte is pumped from theanalyte reservoir totheanalyte
`waste reservoir as in the pinched sample loading except no
`potential is applied to the buffer and waste reservoirs. By not
`controlling the flow of mobile phase in the buffer and separation
`channels, the analyte is free to move into these channels through
`eddy flow, resulting in a more diffuse injection plug.
`When the pinched and floating sample loadings are
`the pinched sample loading is superior in two
`compared,
`temporal stability and plug length. When two or more
`areas:
`analytes with vastly different mobilities are to be analyzed,
`a sample loading with temporal stability ensures that equal
`volumes of
`the faster and slower moving analytes are
`introduced into the separation column. A smaller plug length
`leads to a higher efFiciency and, consequently, to a greater
`component capacity for a given instrument. To determine
`the temporal stability of each sample loading method, a series
`of CCD fluorescence images was collected at 1.5-s intervals
`starting just prior to the analyte reaching the injection cross.
`An estimate of the amount of analyte that is to be injected
`was determined by integrating the fluorescence in the
`intersection and the buffer and separation channels. This
`integrated fluorescence is plotted versus time in Figure 4. For
`the pinched sample loading (circle), a stability of 1% rsd is
`seen, which is comparable to the stability of the illuminating
`laser. For the floating sample loading (square), the amount
`of analyte to be injected into the column increases with time
`because of the flow anisotropy. For a 30-s loading period, the
`volume of the injection plug in the injection cross is ~90 pL
`and stable for the pinched sample loading versus ~300 pL
`and continuously increasing with time for a floating sample
`loading. When the switch is made to the separation mode,
`some of the analyte in the analyte channel and the analyte
`waste channel is injected into the separation column. For the
`pinched sample loading in Figure 3, the volume of the injection
`plug becomes 144 ± 17 pL with a plug length of 158 ± 19
`Mm following the switch to the separation mode. These
`dimensions are estimated from a series of CCD images taken
`immediately after the switch is made to the separation mode.
`The reproducibility for the two injection modes was tested
`by integrating the area of the band profile following intro-
`duction into the separation channel by monitoring the
`
`Analytical Chemistry, Vol. 66, No. 7, April 1, 1994
`
`1109
`
`1500 .it m I
`(a) CCD image of injection cross with no fluorescent analyte
`Figure 3.
`present: CCD images of same region for (b) a pinched sample loading
`and (c) a floating sample loading using rhodamine B.
`
`RESULTS AND DISCUSSION
`The two types of sample loading were tested for sample
`introduction into the separation column. Rhodamine B was
`used as the test analyte for the sample loading studies. The
`analyte is placed in the analyte reservoir and in both injection
`schemes is pumped in the direction of the analyte waste
`reservoir. CCD images of the two types of injections are
`depicted in Figure 3. Figure 3a is a CCD image of the injection
`cross with no fluorescent sample present. Figure 3b shows
`the pinched sample loading prior to being switched to the
`separation mode where the analyte is pumped electrophoret-
`ically and electroosmotically from the analyte reservoir to the
`analyte waste reservoir (left to right) with the buffer from the
`buffer reservoir
`(top) and the waste reservoir
`(bottom)
`traveling toward the analyte waste reservoir (right). The
`brighter (lighter) portions of the image are due to sample
`fluorescence. The voltages applied to the analyte, buffer,
`analyte waste, and waste reservoirs were 90%, 90%, 0%, and
`100% of the power supply output and correspond to electric
`Field strengths in the channels of 270,400,690, and 20 V/cm
`respectively, for an applied potential of 1.0 k V. Consequently,
`the analyte in the injection cross has a trapezoidal shape and
`is constricted and diluted in analyte waste channel (right) by
`
`Agilent Exhibit 1279
`Page 3 of 7
`
`

`

`time ts]
`
`Figure 5. Reproducibility of (a, top) pinched sample loadings and {b,
`bottom) t loatlng sample loadings for five replicate injections of rhodamine
`B using a separation length of 9 mm and a separation field strength
`of 120 V/cm,
`
`separation channel at a point 9 mm from the intersection. For
`five injections with a duration of 40 s, the reproducibility for
`the pinched sample loading (Figure 5a) is 1.7% rsd and for
`the floating sample loading (Figure 5b) is 2.7% rsd. The
`pinched sample loading, not surprisingly, has a higher
`reproducibility because of the temporal stability. With
`electronically controlled voltage switching, the rsd is expected
`to improve for both schemes. The injection plug length and,
`ultimately, the resolution between analytes depend largely on
`both the flow pattern of the analyte and the dimensions of the
`injection cross. For this column, the channel width is ~80
`(im, but a channel width of 10 /im is feasible which would
`decrease the volume of the injection plug to ~-2 pL with a
`pinched sample loading.
`After the sample has been pumped into the cross region
`of the chip, the voltages are manually switched from the sample
`loading to the separation mode of operation.
`In Figure 6 the
`CCD images record the separation process at
`I-s intervals
`with Figure 6a showing a schematic of the section of the chip
`imaged and with Figure 6b-e showing the separation unfold
`in time. Figure 6b again shows the pinched sample loading
`with the applied voltages at the analyte, buffer, and waste
`reservoirs equal. Parts c-e of Figure 6 show the plug moving
`away from the intersection at 1,2, and 3 s, respectively, after
`In Figure 6c the injection plug is
`switching to run mode.
`migrating around a 90° turn, and band distortion is obvious
`due to the inner portion of the plug traveling less distance in
`
`1116 Analytical Chemistry, Vol. 66, No. 7, April 1, 1994
`
`(a) Schematic of region imaged (Injection cross); CCD images
`Figure 6.
`of (b) a pinched sample loading and (c-e) a separation of rhodamine
`B (less retained) and sulforhodamine (more retained) at 1, 2, and 3 s,
`respectively, after switching to the separation mode using a separation
`field strength of 150 V/cm.
`
`the turn than the outer portion. By Figure 6d the analytes
`have separated into distinct bands, which are distorted in the
`shape of a parallelogram.
`In Figure 6e the bands are well
`separated and have attained a more rectangular shape i.e.,
`collapse of the parallelogram due to radial diffusion, an
`additional contribution to efficiency loss.
`When the switch is made from the sample loading mode
`to the separation mode, a clean break of the injection plug
`from the analyte stream is mandatory to avoid tailing. This
`is achieved by pumping the mobile phase from the buffer
`into the analyte, analyte waste, and separation
`channel
`channels simultaneously by maintaining the potential at the
`intersection below the potential of the buffer reservoir and
`above the potentials of the analyte, analyte waste, and waste
`the intersection was
`reservoirs. For
`these experiments,
`maintained at 66% of the potential of the buffer reservoir
`during the run mode. This provided sufficient flow of the
`analyte back away from the injection cross down the analyte
`and analyte waste channels without decreasing the field
`strength in the separation channel significantly. This three-
`way flow is demonstrated in Figure 6c-e as the analytes in
`the analyte and analyte waste channels (left and right,
`
`Agilent Exhibit 1279
`Page 4 of 7
`
`

`

`total = Hinj + Hdet + Hdilt + Hgeo
`
`(1)
`
`where Hmj, H,iet, Hdm, and Hgt0 correspond to the contributions
`to the plate height from the injection plug length, detector
`path length, molecular diffusion in the axial direction, and
`geometry of the column, respectively. The contributions of
`the injection plug length and detector path length are constant,
`the contribution of the axial diffusion depends on the linear
`velocities of the analytes, and the contribution of the column
`geometry depends on the number of turns per unit length in
`the column. The contribution to the plate height from Joule
`heating is neglected because heat dissipation was below 1
`W/m for all experiments.15
`The contributions of the injection plug length and detection
`path length can be calculated by16
`H =
`
`(2)
`
`l2inj/(12L)
`
`H =
`
`l2dJ(12L)
`
`(3)
`
`and Id* are the channel lengths of the injection plug
`where
`and detection path, respectively, and L is the length of the
`separation column between the point of injection and detection.
`Often, these two contributions are negligible compared to the
`other contributions.
`The contribution of the axial diffusion to the band
`broadening follows from the Einstein equation:17
`Him = 2DJu
`
`(4)
`
`where Dm and u are the diffusion coefficient of the analyte
`and the linear velocity of the analyte, respectively. The key
`to minimizing this contribution is performing the separation
`as quickly as possible.
`Less straightforward is the contribution of the geometry
`of the column to the plate height because of the serpentine
`pattern of the column. A geometry such as this serpentine
`enables a significantly greater column length to be employed
`for the separation in a much smaller area (a 165-mm column
`length for the serpentine geometry versus
`~ 10-mm column
`length for a straight geometry in a 10 mm X 10 mm chip
`area). However, by bending the column around 180° turns,
`sacrificing column efficiency for column length becomes a
`concern. We will only consider simple geometrical broadening
`effects here although other contributions such as electric field
`effects are quite feasible, as discussed below. For each turn
`the difference in the length of the column between the inside
`and the outside is
`
`dl = 9 dr
`
`(5)
`
`where 8 is the angle of the turn and dr is the difference in the
`radius of the turn between the inside and the outside of the
`channel, i.e., the channel width. With simple geometrical
`broadening, we assume that the analyte is traveling at the
`
`(15) Monnig, C. A.; Jorgenson, J. W. Anal. Chem. 1991, 63, 802.
`(16) Sternberg, J. C. Adv. Chromatogr. 1966, 2, 205.
`(17) Giddings, J. C. Dynamics of Chromatography, Part I: Principles and Theory,
`Marcel Dekker: New York, 1965; Chapter 2.
`
`Analytical Chemistry, Vol. 66, No. 7, April 1, 1994
`
`1111
`
`time [a]
`Figure 7. Electropherograms at 33 (top), 99 (middle), and 165 mm
`(bottom) from the point of injection for rhodamine B (less retained) and
`sulforhodamine (more retained) using a separation field strength of
`170 V/cm and pinched sample loading. The electropherograms have
`been offset vertically to facilitate viewing.
`
`Figure 8. Variation of the plate number with separation channel length
`for rhodamine B (circle) and sulforhodamine (square) with best linear
`fits (line) using a separation field strength of 170 V/cm and pinched
`sample loading.
`
`respectively) move farther away from the intersection with
`time. Three-way flow permits well-defined, reproducible
`injections (Figure 5) with minimal bleeding of the analyte
`into the separation channel.
`To obtain electropherograms in the conventional manner,
`single-point detection with the helium/neon laser was used at
`different locations down the axis of the separation column.
`The efficiency at 10 evenly spaced positions was monitored,
`each constituting a separate experiment. Figure 7 depicts
`selected electropherograms at 33, 99, and 165 mm from the
`point of injection.' The efficiency data are plotted in Figure
`8. At 165 mm from the point of injection, the efficiencies of
`rhodamine B and sulforhodamine are 38 100 and 29 000 plates,
`respectively. Efficiencies of this magnitude are sufficient for
`many separation applications. The linearity of the data
`provides information about the uniformity and quality of the
`If a defect in the
`channel down the length of the column.
`channel, e.g., a large etch pit, was present, a sharp decrease
`in the efficiency would result. This is not the case.
`The primary contributions to band broadening for these
`experiments are shown in the following equation for the total
`plate height:
`
`Agilent Exhibit 1279
`Page 5 of 7
`
`

`

`same velocity on both the inside of the column and the outside
`of the column through the turn. Consequently, the shift or
`distortion in the band profile is equivalent to d/ in eq 5.
`For the 90® turn immediately following the injection cross
`in the separation channel (Figure 6a), 6 and dr are equal to
`7r/2 and 90 pm, respectively, and consequently, dl is 140 pm,
`which should be the contribution to band broadening for this
`one turn. From the experimental data in Figure 6d, the shift
`in the band shape is calculated from the center of the bands
`for each row of pixels from this CCD image, and the band
`distortion is estimated to be 150 pm. The slight discrepancy
`(7%) can be attributed to the nonrectangular injection plug.
`The geometry of the turns could also produce an additional
`distortion due to spatially varying electric field strengths within
`the turn. Thus, an ion on the inside of the turn would
`experience a higher electric field than an ion on the outside
`of a turn, resulting in a greater linear velocity for the ion on
`the inside. Coupled with the difference in the lengths of travel,
`an ion on the inside of a turn and an ion on the outside would
`be separated by a distance 2d/ following a turn, e.g. 280 /am
`for a 90® turn. This distortion is too large compared to the
`experimentally measured band distortion of 150 pm observed
`in Figure 6d.
`Consequently, the contribution to the plate height equation
`due to the turns in the serpentine pattern of the separation
`column is written as follows:
`
`Hgeo = n(w6)2/(l2L)
`
`(6)
`
`where n is the number of identical turns, w is the width at the
`top of the channel, and 6 is the angle of a single turn. The
`transit time between turns is assumed to be long compared
`to the time to diffuse across the channel. Therefore the position
`of an individual molecule in the cross section of the channel,
`from turn to turn, is random, and Hgeo is a function of n rather
`than n2.18 Assuming the number of turns per unit distance
`then Hgeo will be constant.
`Importantly, this
`is constant,
`contribution to the plate height decreases as the square of the
`channel width.
`The contributions from injection plug length, detector path
`for a given
`length, and column geometry are constant
`experiment while the contribution from the axial diffusion
`In Figure 9, the
`decreases with increasing linear velocity.
`linear velocity data demonstrate an
`plate height versus
`increasing efficiency with increasing linear velocity. The three
`constant terms are combined, added to an axial diffusion term,
`and fitted to the experimental data. The sum of the three
`constant contributions for rhodamine B and sulforhodamine
`respectively, and the diffusion
`are 4.02 and 5.39 pm,
`coefficients, Dm, are 3.27 X 10-6 and 3.33 X 10-6 cm2/s,
`respectively.
`The individual constant plate height contributions for this
`experiment are estimated. The injection plug length is equal
`from eq
`to 300 pm for the the injection in Figure 6, and
`2 is 0.23 pm. Since the detection is on-column, the detector
`path length is equal to the laser spot size, which is ~ 100 pm.
`Because the
`to 25 nm.
`Therefore, Hdet
`is equal
`(eq 3)
`separation was monitored at a distance 33 mm from the point
`
`(18) Giddings, J. C. J. Chem. Educ. 1958, 35, 588.
`
`1112 Analytical Chemistry, Vol. 66, No. 7, April 1, 1994
`
`Figure 9. Variation of the plate height (H) with linear velocity (u) for
`rhodamine B (circle) and sulforhodamine (square) with best fits of eq
`1 (lines) using a separation length of 33 mm and pinched sample
`loading.
`
`of injection, the separation column includes one 90® turn
`(described above) and four 180° turns. Using eq 6, Hgeo is
`equal to 0.85 /am.
`The fitted constant values from the data in Figure 9,4.02
`for rhodamine B and 5.39 for sulforhodamine, are not equal
`and are both greater than the sum of the three estimated
`constant contributions of 0.90 pm. Limitations of the present
`apparatus prevented the use of field strengths greater than
`170 V/cm, and therefore, more comprehensive data could not
`be generated.
`
`CONCLUSIONS
`The use of microchip devices for liquid-phase separations
`and analysis appears to be quite promising. Several important
`aspects for improving the performance of these devices were
`discussed in this work. Cold bonding of cover plates to enclose
`micromachined channels will allow the use of a broad range
`of substrate materials and could lead to greater device yields.
`An injection scheme was presented that provides high
`performance with respect to accuracy, precision, and small
`volume while remaining simplistic with respect to fabrication.
`The device used in these studies has a smaller area/column
`length ratio than previously reported due to the use of a
`serpentine column geometry.6-9 The use of such geometries
`will be necessary to move microchip liquid-phase analysis
`devices into the sensor
`realm. Band-broadening phenomena
`associated with the serpentine structure, while measurable,
`do not appear to be a severely limiting problem for capillary
`electrophoresis implementations employing field strengths less
`than ~200 V/cm. The plate height associated with the
`serpentine geometry was experimentally measured and found
`to be approximately what would be expected for path length
`differences associated with the turns. This additional plate
`1 pm for the 165-mm column
`height was calculated to be ~
`length. Reduction in channel widths should allow reduction
`of this effect to an acceptable level.
`
`ACKNOWLEDGMENT
`This research was sponsored by the U.S. Department of
`Energy (DOE), Office of Research and Development. Oak
`Ridge National Laboratory is managed by Martin Marietta
`
`Agilent Exhibit 1279
`Page 6 of 7
`
`

`

`Energy Systems, Inc., for the U.S. Department of Energy
`under Contract DE-AC05-840R21400. 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, for L.B.K. to the DOE
`Laboratory Cooperative Postgraduate Research Training
`Program, and for R.H. to the ORNL postdoctoral research
`associate program. These programs are administered by the
`
`Oak Ridge Institute for Science and Education and ORNL.
`The authors acknowledge many useful discussions with Drs.
`Roswitha S. Ramsey and Georges Guiochon.
`
`Received for
`1994.®
`
`review October 5, 1993. Accepted January 10,
`
`'Abstract published in Advance ACS Abstracts, February IS, 1994.
`
`Analytical Chemistry, Vd. 66, No. 7, April 1, 1994
`
`1113
`
`Agilent Exhibit 1279
`Page 7 of 7
`
`