Anal. Chem. 1994,66, 2369-2373
Open Channel Electrochromatography on a Microchip Stephen C. Jacobson, Roland Hergenriider, Lance B. Koutny, and J. Michael Ramsey' Chemical and Analytical Sciences Division, Oak Ridge National Laboratoty, Oak Ridge, Tennessee 3783 1-6 142
A glass microchip having a channel with a cross section of 5.6 pm high and 66 pm wide was fabricated using standard
photolithographic and etching techniques. The surface of the channel was chemically modified with octadecylsilane to function as the stationary phase for open channel chromatography. Electroosmotic flow was used to "load"the sample into the microchip and to "pump"the mobile phase during the experiments. For electric field strengths in the separation column from 27 to 163 V/cm, the linear velocity for the electroosmotic flow ranged from 0.13 to 0.78 mm/s. Detection was performed using direct fluorescence for separation monitoring and indirect fluorescence for void time measurements. Plate heights as low as 4.1 and 5.0 pm were generated for unretained and retained components, respectively. Compact, reliable, and inexpensive instrumentation is desirable for a number of chemical measurement problems including field surveys, on-line process control, and remote sensing. One approach to this problem is the microfabrication of conventional chemical instrumentation. Micromachining using standard lithographic and etching techniques is one method for fabricating these miniature devices.' Chemical separation devices appear to be particularly amenable to microfabrication. Micromachined substrates have been produced for gas chromatography,2 liquid ~hromatography,~ and capillary electroph~resis.'~ In the case of liquid chromatography, no experimental demonstration has been presented. Liquid chromatography is a common method for separating and analyzing neutral species. Implementing liquid chromatography on a microchip simply requires packing of the separation channel with a separation phase or chemically modifying the channel wall for open channel operation. The latter approach was taken here. The reduced dimensions of microchip separation devices have the potential advantage that the contribution of the resistance to mass transfer decreases as the dimensions of the channel decrease.1° (1) Manz, A.; Fettinger, J. C.; Verpoorte, E.;Liidi, H.; Widmer, H. M.; Harrison, D. J. Trends Anal. Chem. 1991,10, 144. (2) Terry, S. C.; Jerman, J. H.; Angell, J. B. IEEE Trans. ElecfronDevices 1979,
26, 1880.
(3) Manz, A.;Miyahara, Y.;Miura, J.; Watanabe, Y.; Miyagi, H.;Sato, K.Sens.
Actuators 1990,Bl, 249.
(4) Manz, A.; Harrison, J.; Verpoorte, E. M. J.; Fcttingcr, J. C.; Paulus, A,; Lildi, H.; Widmer, H. M. J . Chromafogr. 1992,593,253. (5) Harrison, D. J.; Manz, A.; Fan, 2.;Lildi, H.; Widmer, H. M. Anal. Chem.
1992,64,1926. (6) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993,65,1481. (7) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, 2.;Effenhauser, C. S.; Manz, A. Science 1993.261,895. (8) Jacobson, S. C.; HergenrMer, R.; Koutny, L. B.; Warmack, R. J.; Ramscy, J. M. Anal. Chem. 1994,66,1107. (9) Jacobson, S. C.; HergenrMer, R.; Koutny, L. B.; Ramsey, J. A. Anal. Chem. 1994,66,11 14. 0003-2700/94/03662389$04.50/0
0 1994 American Chemical Soclety
Consequently, shorter columns and higher mobile-phase velocities can be used, resulting in shorter analyses times. An important issue to resolve is the method by which to pump the mobile phase during the experiments. The technically simplest method to implement on the microchip substrate is electroosmosisllJ2 because no moving parts are required. In capillary liquid chromatography, electroosmotic "pumping" of the mobile phase has been demonstrated for both packed13J4 and open15capillaries. By pumping the mobile phase with electroosmosis, the flow profile of the mobile phase is nearly flat. Conversely, pressure-driven capillary liquid chromatography has a Poiseuille flow profile, and consequently, the resistance to mass transfer for the mobile phase is approximately 2 times higher for a pressure-driven system than an electroosmotically driven system.16 Experimental verification of this has been dem0n~trated.l~ The benefits of the electroosmotic flow also are seen in the manipulation of the sample during injection on a microchip. By loading the sample onto the column with electroosmotic flow, valveless, timeindependent, nonbiased injections can be made with high reproducibility.8 In the case of open tubes, as the diameter of the capillary decreases, the resistance to mass transfer for the mobile phase decreases, but other problems arise. The amount of sample that can be injected without overloading the column is proportional to the surface area of the capillary and may fall below the limits of detection. Detection for path length dependent schemes becomes instrumentally prohibitive. Also, injection and detection volumes must be scaled proportionally to prevent excessive contributions to band broadening. One solution is to fabricate columns that have a high aspect ratio, i.e., channel width to channel height. This affords improved mass transfer in one dimension while the sample load, detection path length, and detection volume are maintained in the other dimension. For a flat flow profile comparable plate heights between a capillary and a channel with a high aspect ratio are obtained if the height of the channel is approximately 0.6 times the diameter of the ~api1lary.l~Using field flow fractionation (FFF) technology, channels with aspect ratios up to 1500 have been fabricated, chemically modified, and used for gas18 and liquid ~ h r o m a t o g r a p h y ~ ~ ~ 9 ~ 0 (10) Golay, M. J. E. In Gas Chromatography; Dwty, D. H., Ed.;Butterworths: London, 1958; p 36. (11) Burgreen, D.; Nahche, F. R. J . fhys. Chem. 1964, 68,1084. (12) Rice, C. L.; Whitehead, R. J . fhys. Chem. 1965,69,4017. (13) Jorgenson, J. W.; Luhcs, K. D. J . Chromafogr. 1981,218,209. (14) Knox, J. H.; Grant, 1. H. Chromotographia 1987,24, 135. (15) Tsuda, T.; Kazuhiro, N.; Nakagawa, G. J. Chromatogr. 1982,248,241. (16) Martin, M.; Guiochon, G. Anal. Chem. 1984,56,614. (17) Giddings, J. C. J . Chromafogr.1%1, 5,46. (18) Martin, M.; Jurado-Baizaval. J.-L.; Guiochon, G. Chromatographto 1984, 16,98.
Ana~caIChemIstry,Vol. 66,No. 14, July 15, 1994 2369
la
b
+
7
manual switches
microchip
laser I
HV power supply
2.2 MR
-4 sample loading mode
-0
-
separation mode
W
10 mm
substrate
In this paper, a microchip was fabricated to perform electrochromatography in open channels. Laser-induced fluorescence was used to monitor the separationsdirectly and thevoid time indirectly. The mechanisms for band broadening are discussed, and calculated values for flat flow profiles are compared to experimental values.
EXPERIMENTAL SECTION The channels on the glass microchip substrate were fabricated using standard photolithographictechniques and chemical wet etching.21 The micromachined substrate and cover plate were joined using a direct bonding technique as described previously.* Figure l a shows a schematic of the column design implemented for the experiments. The serpentine column geometry has enclosed column lengths of 6.4 mm from the buffer reservoir to the injection cross, 9.4 mm from the analyte and analyte waste reservoirs to the injection cross, and 17 1 mm from the waste reservoir to the injection cross. The serpentine column design enables a long column length to be fabricated within a compact area. On this microchip a separation column length of, 165 mm is fabricated within a 8 mm X 8 mm area. Only an 11-mm straight separation column design could be fabricated in the same space. 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 (19) Manin, M.; Jurad+Baiival, J.-L.; Guiochon, 0. C.R.Acd. Sc. Parfs,Ser. II 1981, 295, 519. (20) Giddinga. J. C.; Ckng. J. P.; Myers, M.N.; Davis, J. M.;Caldwell, K. D. 1. Chromrogr. 1983, 255, 359. (21) For example: KO,W. H.; Suminto, J. T. In Sensors; Gopcl, W., H w ,J., Zcnnel. J . N., Eda.;VCH: Weinhein, Germany, 1989; Vol. 1, pp 107-168.
2370 Arrp&thl
m w , Val. 68,No. 14, Julv
15, 1884
geometry is trapezoidal. Figure 2 shows the profile of the channel cross section with dimensions of 5.6 pm high and 72 pm wide at the top and 60 pm wide at the bottom. The dimensions were obtained using a profilometer (Alpha-Step 200, Tencor Instruments). The aspect ratio for the channel is
6 = wo.dh
(1)
where ~ 0 . 5is the channel width at half-height and h is the channel height. From the channel profile in Figure 2, w0.s and h are 66 and 5.6 gm, respectively, corresponding to an aspect ratio of 12. After bonding the cover plate to the substrate and thereby enclosing the channels, the rmersc-phase coating was chemically bonded to the walls of the channels.22 First, the waste reservoir was affixed to the substrate via epoxy to act not only as a reagent vessel but also as a connection to a helium supply. The column was treated with 1 M sodium hydroxide and then rinsed with water. The column was dried at 125 O C for 24 h while being purged with helium at a gauge pressure of (22) For example: J"n,
J. W.;Guthrie, E. J. 1.Chromcrtogr. 1983,255,335.
approximately 50 kPA. A 25% (w/w) solution of chlorodimethyloctadecylsilane (ODS, Aldrich) in toluene was loaded into the waste reservoir and pumped into the channel with an overpressure of helium at approximately 90 kPa. The ODs/ toluene mixture was pumped continuously into the column throughout the 18-h reaction period at 125 OC. The channel was rinsed with toluene and then with acetonitrile to remove the unreacted ODs. The separations were monitored on-microchip via fluorescence using an argon ion laser (35 1.1 nm, Coherent Innova 90) for excitation and a photomultiplier tube (PMT, Oriel 77340) to collect the fluorescence signal. The PMT, with collection optics, was situated below the microchip with the optical axis perpendicular to the microchip surface. The laser wasoperated at approximately 20 mW, and the beam impinged upon the microchip at a 45O angle from the microchip surface and parallel to the separation channel. The laser beam and PMT observation axis were separated by a 135' angle. The power supply (Spellman CZE1000R) for the electrophoresis was operated between +0.63 and +3.8 kV relative to ground. Figure 1b shows a diagramof high-voltageswitching apparatus and detection/data acquisition system. The high-voltage switches and leads were interlocked to prevent electrical shock. Platinum electrodes were inserted into the reservoirs for electrical contact. Higher voltages could not be utilized due to limitations of the manual switching apparatus (4 kV). The analytes used for the experiments were coumarin 440 (C440), coumarin 450 (C450) and coumarin 460 (C460, Exciton Chemical Co., Inc.) at 10 pM for the direct fluorescent measurements of the separations and 1 pM for the indirect fluorescent measurements of the void time. A sodium tetraborate buffer (10 mM, pH 9.2) with 25%(v/v) acetonitrile was the buffer in all experiments. Due care must be exercised with the Coumarin dyes because the chemical, physical, and toxicological properties have not been fully i n ~ e s t i g a t e d . ~ ~ The microchip was operated under a "pinched sample loading" mode and a "separation" mode as described previously.* The sample is loaded into the injection cross via a frontal chromatogram traveling from the analyte reservoir to the analyte waste reservoir, and once the front of the slowest analyte passes through the injection cross, the sample is ready to be analyzed. The analyte flowing through the injection cross is both defined and confined by electroosmotic pumping of the buffer from the buffer and the waste reservoirs toward theanalyte waste reservoir. Toswitch to theseparation mode, the applied potentials are merely reconfigured by manually throwing a switch. Now the primary flow path for the separation is from the buffer reservoir to the waste reservoir. In order to inject a small analyte plug into the separation column and to prevent bleeding of the excess analyte into the separation column, the analyte and the analyte waste reservoirs are maintained at 57% of the potential applied to the mobilephase reservoir. This three-way flow forces the excess analyte to pull away from the injection cross and prevents analyte from bleeding into the separation column. This method of loading and injecting the sample is time-independent, nonbiased, and reproducible. A separation length of 58 mm was used for all experiments. At this distance the analytes were baseline resolved for all (23) Exciton Chemical Co., Inc.
0
50
100
160
200
time [SI Figure3. Indirectchromatogramof H20for voM volumn measurement with u = 0.65 mmls.
t
0
'I
c450
60
100 time
1
/I
150
200
[SI
Figure 4. Chromatogram of C440, C450 and C460 with u = 0.65 mm/s.
linear velocities, and the analysis times were not excessive. The void times were estimated indirectly by having C440 and C450 in the mobile phase and injecting a water plug. The composition of the injection plug therefore was 5 mM sodium tetraborate with 25%acetonitrile. RESULTS AND DISCUSSION An indirect chromatogram of water at a linear velocity of 0.65 mm/s is depicted in Figure 3. Indirect fluorometry was a simple method for determining the mobile-phase velocity which was pumped electroosmotically. The presence of the low concentration of coumarins in the mobile phase was assumed not to affect the electroosmotic velocity. The highest linear velocity was 0.78 mm/s for an electric field strength in the separation column of 162 V/cm. This value compares well with the l i t e r a t ~ r eand ' ~ corresponds to a volumetric flow rate of 0.3 nL/s. In Figure 4, a chromatogram of the coumarins is shown for a linear velocity of 0.65 mm/s. The retention ratios and efficiency data from this separation are listed in Table 1. For C440, 11 700 plates were observed, which corresponds to 120 plates/s. The most retained componeIit, C460, has an efficiency nearly 1 order of magnitude lower than C440, 1290 plates. The undulating background in the chromatograms is due to background fluorescence from the glass substrate and shows the power AnaIflicalChemistry, Vol. 66, No. 14, July 15, 1994
2371
T-1. R U ~ i ~ U o n n d ~ W t o r a W U t l U W U W h FlgIua9md4 compound R N H (rm) 1.o 0.91 0.79 0.64
H20
C440 C450 C460
13800 11700 2950 1290
4.2 5.0 19.7 44.8
because the film thickness formed from m o " s i l a w s small relative to the c h a d diannsions.x
is
The plate heights for t& injection plug length, detector obervation length?' and column g c o m e w are constant contributions for a given experiment and can be written Hw = 12@/( 1 2 L 4
0.8
(4)
Hda = l2,,J(l2&)
0.6
where lhj is the length of the injection plug, l& is the detector obsarvationlength,L,is thesoparationlength,nis thenumber of turns, w is the width of the channel, and B is tho angle of the turn. The band distortion due to a tam in the serpentine column geometry has been observed experhatally to be equal to the difference in the path lengths between the inside and outside of the channel.' Other phenomena such as electric field effects or eddy flow have not been observdd, although thtse contributions are quite feasible. The vehcity-dependent terms of the plate height arc axiai diffusion and resistance to mass transfer to the mobile phase. For axial diffbion, the expression isu
a 09
Hdilr= 2DJu
instability of the laser. This, however, did not hamper the quality of the separations or detection. A compilation of the separations is plotted in Figure 5 as the variation of the linear velocities with the electric field strength in the separation channel. This figure demonstrates that over the span of time taken to perform the separations, no change in the column dynamics was observed; Le., the bonded phase was stable. Also, the separation selectivity, a,can be calculatedz4 ajj = (rRj
- t&/(tw-
(2)
10)
where t R is the elution time of a retained molecule, to is the elution time of an u n r e t a i d marker, i designates a less retained molecule, and j designatesa more retained molecule. ) O2.44 f 0.13 and C Y C W For the data in Figure 5 , Q C ~ ~ , C ) = = 2.19 i 0.10. This demonstrates that the separation selectivity is independent of the separation field strength. The most important measure of a separation system is the plate height. The significant contributions to the total plate height for this system are H,,,, = Hhi + Ha
+ Hw+
H M + H,
(3)
where Hhj is from the injaction plug length, H a is from the detector observation length, H-is from the column geometry, Hdiffis from axial diffusion, and Hm is from mass transfer for the mobile phase. The contribution to the plate height from Joule heating is considered negligible for these experiments because the power dissipatd was well below 1 W / m z and from resistance to mass transfer for the,stationary phase B. L.;Snyder, L. R.; H m a t h , C. AR Introductlon S c f e m , John Wiley and Sonr: New Yorf 1973.
(24) Kargw.
IO
Sepation
(25) Monnig, C. A,; J o r g m , J. W. AM/. C h . 1991.63, 802.
2372
A M & i k ~ l m & y Vd. , 66, Ab. 14, 3rJL 15, 1004
(7)
where Dm is the diffusion coefficient of the analyte in the mobile phase and u is the linear velocity of the mobile phase. The resistance to mass transfer for the mobile phase with infinitely parallel plates and plug flow is given by17 Hmc = C-u = 'l6(l- R)zh2/D,u
(8)
where R is the retention ratio, Le., the void time, to, divided , h is the height by the retention time of the analyte, t ~and of the channel. The analogous expression for a capillary of diameter, d, is" (9) , ~ ~
The ratio of eqs 8 and 9 is
Under conditions where the mass-trader term dominates, comparable plate heights arc produced by rectangularchannels and capillaries when h = 0.U. Consequently, with a rectangular chaand geomcrtry havinga high aspect ratio, high efficiency is feasible without sacrificing detection path length or injection and detection volumcs. The constant contributions to the plate height for these experiments are calculated. The length of the injection plug is approximately 300 pm,8 which corresponds to Hbj = 0.13 pm. The detection is on-microchip, and the channel length (26) KJIOX,J. H. 1.Chromatogr. Sei. I-, 18,453. (27) Sternba& J. C. A&. .l%6,2.205. (28) G ~ J . C . D y M m l u o f C ~ ~ h y m, dP p~l el s a m i T ~ , Marcel Dekker: New Yorf 1965; Chapter 2.
75
--t
50
c480
o ~ " " ' " " " ' " " ' ~ 0
0.3
0.6
u
0.9
["/SI
Figure 6. Variation of the plate height (H) with the linear velocity (4 for H20, C440, C450, and C460. Lines are eq 11 fltted to the experimental data. Table 2. FHted and Calculated Plate Helghl Coeffklenlr for Figure 6 compound D, (mm2/s) C m (s) Cm.h" (s)
H2O C440 C450 C460
9.7 x lo" 1.0 x 10-3 1.4x 10-3 1.6 x 10-3
1.9 X 10-3 2.4 X le2 6.4X 1P2
4.2x 10-5 2.3 X lo" 6.8 X 10-4
Equation 8.
illuminated is equal to the laser spot size. Thus, ldct = 100 pm,andHdct= 0.014pm. Thecontribution to theplate height from the column geometry is Hgeo = 0.53 pm, which is calculated for one turn with an angle ?r/2 plus seven turns with an angle ?r and width of the channel at the top equal to 72 pm. The sum of all the constant contributions is 0.67 pm. The experimental plate height data are plotted versus the linear velocity of the mobile phase in Figure 6. The following equation is fitted to these data:
H,,,= 2D,/u
+ C,u + 0.67 pm
(11)
with the determined coefficients listed in Table 2. The constant, 0.67 pm, is taken from experimental parameters rather than being determined in the fitting procedure to lend consistency to the values D, and C,. The values of the diffusion coefficients are slightly higher than expected (0.001 mm2/s); however, the number of data points in the low velocity region is few. Equation 8 assumes infinitely parallel plates or, from a practical standpoint, rectangular channels with a large aspect ratio, e.g., 6 > 400, whereas B = 12 for this experiment. The calculated values for Cm,h with a channel height of 5.6 pm
with D, = 0.001 mmz/s are listed in Table 2. The experimental values are 2 orders of magnitude larger than the calculated values for the channel. This difference may be attributed to the trapezoidal geometry of the channel cross section (Figure 2). The probability of a molecule contacting the bonded phase in the corners of the channel is higher than in the rest of the channel. If this is the case, then the band broadening would increase with increasing retention as observed. The cross section of the channel can be divided into three regions approximated by a right triangle on each end and a rectangle in the center. With this simple picture, the surface-to-volume ratio is 0.81 mm-l for the triangular regions and 0.33 mm-l for the rectangular region. Consequently, the velocity distribution of retained molecules for the trapezoidal channel geometry would be broader than in the case of infinitelyparallel plates, leading to band broadening. The extent of this contribution is under further investigation. In conclusion, the prospect of microchip devices for the separation of neutrals using electroosmotic pumping is promising. The band-broadening effects of the trapezoidal channel cross section on the separations require more study in order to improve the efficiencies. Ultimately, microchips with micromachined channels having a height of 1 pm and a width of 50 pm could offer the benefits of small contributions from resistance to mass transfer for the mobile phase with reasonable values for the injected volume, detection path length, and detection volume. The contribution of the serpentine column geometry to the total plate height ranges from 1 to 13%,depending on the analyte and the linear velocity of the mobile phase. This contribution can be minimized by decreasing the width of the channel, the angle of the turns, and the number of turns per unit length. 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 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 theU.S. DOE, for L.B.K. to the DOELahratory Cooperative Postgraduate Research Training Program, 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. The authors acknowledge many useful discussions with Drs. Roswitha Ramsey and Georges Guiochon. Received for review Janurary 4, 1994. Accepted April 14, 1994.@ *Abstract published in Advance ACS Abstracts, May 15, 1994.
Analytcal Chemlstry, Vol. 66, No. 14, Ju& 15, 1994
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