Generating Electrospray from Microchip Devices Using Electroosmotic

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Anal. Chem. 1997, 69, 1174-1178

Generating Electrospray from Microchip Devices Using Electroosmotic Pumping R. S. Ramsey and J. M. Ramsey*

Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6142

A method of generating electrospray from solutions emerging from small channels etched on planar substrates is described. The fluids are delivered using electroosmotically induced pressures and are sprayed electrostatically from the terminus of a channel by applying an electrical potential of sufficient amplitude to generate the electrospray between the microchip and a conductor spaced from the channel terminus. No major modification of the microchip is required other than to expose a channel opening. The principles that regulate the fluid delivery are described and demonstrated. A spectrum for a test compound, tetrabutylammonium iodide, that was continuously electrophoresed was obtained by coupling the microchip to an ion trap mass spectrometer. Miniaturized chemical instruments that incorporate liquidphase separations such as electrophoresis and electrochromatography are being increasingly recognized as a convenient means of manipulating and analyzing small quantities of material.1-15 These “micro” devices are fabricated on glass or quartz substrates using standard micromachining techniques such as photolithography, wet chemical etching, and thin-film deposition .16 The products are planar devices with micrometer-sized channels through which materials are manipulated using electrokinetic forces. Structures with various interconnecting channels may be easily fabricated, allowing separations and reactions to be performed on picoliter volume samples at relatively high speed. Perhaps the greatest promise of these devices lies in the ability (1) Manz, A.; Harrison, D. J.; Verpoorte, E.; Widmer, H. M. Adv. Chromatogr. 1993, 33, 1. (2) Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Lu ¨ di, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253. (3) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895. (4) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114. (5) Jacobson, S. C.; Ramsey, J. M. Electrophoresis 1995, 16, 481. (6) Jacobson, S. C.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1995, 67, 2059. (7) Burggraf, N.; Manz, A.; Effenhauser, C. S.; Verpoorte, E.; de Rooij, N. F.; Widmer, H. M. J. High Resolut. Chromatogr. 1993, 16, 594. (8) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2858. (9) Effenhauser, C. S.; Paulus, A.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2949. (10) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11348. (11) Woolley, A. T.; Mathies, R. A. SPIE Proc. 1995, 2398, 36. (12) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 2369. (13) Moore, A. W., Jr.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1995, 67, 4184. (14) Jacobson, S. C.; Hergenro ¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127. (15) Jacobson, S C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720. (16) Ko, W. H.; Suminto, J. T. In Sensors; Gopel, W., Hasse, J., Zemmel, J. N., Eds.; VCH: Weinhein, Germany, 1989; Vol. 1, pp 107-168.

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to design and construct systems with the necessary geometry to perform diverse and complex chemical and analytical functions on a given sample.14,15,17 Reduced sample and reagent consumption and increased precision and reproducibility, relative to benchscale instruments, are other advantages that have been cited.4,17-19 Analyte detection remains an important issue for the microchip devices, however, given the small sample volumes typically analyzed. Most analyses have, in fact, been conducted using laserinduced fluorescence (LIF), where small sample volumes are easily probed and high sensitivity in terms of low mass detection limits may be obtained. Single-molecule detection has been accomplished “on-microchip” using LIF.20 To extend the applicability of these miniaturized devices to species that are not fluorescent (or are easily converted to fluorescent species), it is desirable to have other modes of detection available. The informational output would be significantly improved by coupling these devices, for example, with mass spectrometry (MS) where both molecular weight and structural data for analytes may be acquired. We report here a method for delivering solutions to a channel opening and for generating electrospray from solutions emerging from the channel opening, enabling these microdevices to be directly interfaced with the macroworld of mass spectrometers. EXPERIMENTAL SECTION The microchips were fabricated using standard procedures as previously described.6 Figure 1 shows a schematic diagram of a planar glass microchip used to generate electrospray. The drawing is not to scale. The relative channel lengths for the experiments discussed below are 33 mm from the uppermost reservoir to the side-arm intersection, 22 mm for the side arm, and 1 mm from the side-arm intersection to the channel opening. The channels were machined into the surface of the glass substrate using photolithographic patterning and wet chemical etching. Channel dimensions were ∼10 µm deep and 60 µm wide at half-depth. A glass cover plate was then direct bonded over the open channel structure to form a closed network. Small fluid reservoirs attached with epoxy resin where the channels exit from underneath the cover plate allow fluidic communication and electrical contact with the channels. The channel opening from where the electrospray is generated was created by scoring and breaking the fabricated microchip. The exit end on some (17) Jacobson, S. C.; Koutny, L. B.; Hergenro¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472. (18) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993, 65, 1481. (19) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M., Anal. Chem. 1994, 66, 1107. (20) Fister, J. C.; Davis, L. M.; Jacobson, S. C.; Ramsey, J. M. High Sensitivity Detection on Microchips. Presented at Laser Applications to Chemical Analysis, Orlando, FL, March 18, 1996. S0003-2700(96)01067-0 CCC: $14.00

© 1997 American Chemical Society

Figure 1. Schematic diagram of a microchip used to electroosmotically pump fluids and to generate electrospray.

microchips were polished to allow a clear microscopic view of the opening and liquid as it emerged from the channel. The polishing did not noticeably affect microchip performance. Electrical potentials were applied to the solutions in the top and sidearm reservoirs through platinum wires connected to separate power supplies (Spellman CZE1000R). A linear polyacrylamide coating was used to eliminate or reduce electroosmotic flow in the side-arm channel.21 All channels were initially rinsed with 0.1 N sodium hydroxide and water. The side-arm channel was then filled and flushed with a 1% (v/v) solution of 3-(trimethoxysilyl)propyl methacrylate (Aldrich) in methanol for a minimum of 30 min by applying vacuum to the exit port. The channel was then rinsed with methanol and flushed for 30 min with a 3% (w/v) aqueous acrylamide solution containing 0.1% (w/v) potassium persulfate (Bio-Rad Laboratories) and 0.1% (v/v) N,N,N′,N′- tetramethylethylenediamine (Bio-Rad Laboratories). Excess acrylamide solution was removed and the channel extensively rinsed with water. Reservoirs other than the side arm were filled with methanol during treatment with methacrylate and with water during treatment with acrylamide. The microchips were imaged using laser-induced fluorescence to monitor fluid flow. The beam from an argon ion laser (514.5 nm, 100 mW) was expanded to a 4 mm spot on the surface of the microchip using a lens, and the fluorescence signal was collected with an optical microscope following spectral filtering (550 nm cut-on) and measured with the CCD. Rhodamine B and 6G dye were used for imaging. Mass spectrometry was performed on a Finnigan MAT ITMS (San Jose, CA) modified for ES ionization.22 The microchip was secured with adhesive to a flat plate which was bolted to an x-y-z translational stage. The chip extended ∼1 cm past the edge of the plate, and the channel opening was carefully positioned 3-5 mm from the inlet orifice on the front aperture plate of the mass spectrometer. Focusing conditions were optimized to maximize the intensity of the analyte ions. Ions were injected for 100 ms and held for an additional 300 ms in the ion trap to promote desolvation prior to mass analysis. (21) Hjerten, S. J. J. Chromatogr. 1985, 347, 191. (22) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284.

DISCUSSION As a solution-based technique, a variety of electrospray ionization sources have evolved to accommodate different methods of liquid sample introduction including electrically driven flow.23-26 Successful coupling of capillary electrophoresis (CE) with MS requires that the interface supply the conditions necessary to establish a stable electrospray and the electrical bias necessary to drive the electrophoresis. In addition, the efficiency of the CE separation should not be impaired by the interface. Interconnected channel structures that allow fluidic and electrical contact with a CE channel are easily fabricated in planar structures and allow flexible designs for accomplishing electrospray CE. The planar substrates onto which the capillary channels are etched, however, differ significantly from other electrospray ion sources where fluids are sprayed from needles or capillary tubes.26-28 To determine whether solutions emerging from a channel could be electrosprayed without modifying the exit end of the microchip, some initial experiments were performed using positive pressure to force fluid through the channel exit. In general, the low currents generated in electrospray are insufficient to induce the electroosmotic flow necessary to supply the fluid to the channel opening to sustain an electrospray. Pressures of a few psig were therefore applied to the uppermost reservoir of a simple crossmicrochip (similar in structure to that illustrated in Figure 1 but without the side arm) while a potential of 3-6 kV, relative to a grounded electrode, was also applied to the reservoir. The electrode (a flat aluminum disk) was placed ∼3-5 mm from the exit of the microchip. Parts a and b of Figure 2 are respectively photomicrographs showing a bead of liquid as it emerges from the channel and its deformation into a Taylor cone by the electric field between the droplet and the ground plate as a potential is applied. A fine spray can also be seen exiting the tip of the Taylor cone. The current measured by a picoameter at the target electrode ranged upward from 20 nA, depending upon the spacing from the microchip, the composition of the solution, and the applied potential. Pure water and mixtures of water and methanol were electrosprayed in this manner. An alternative method of transporting fluid to the channel opening is to use electroosmotically induced pressures. The velocity, v, of a fluid through a channel when driven by electroosmosis is given by eq 1, where E is the electric field strength, ζ is

v ) oζE/4πη

(1)

the zeta potential across the Stern layer at the channel-fluid interface,  is the dielectric constant of the fluid, ο is the permitivity of free space, and η is the viscosity of the fluid at the interface. The steady-state velocity of the electroosmotically induced fluid flow does not depend on the cross-sectional dimensions or geometry of the channel when all dimensions are much (23) Niessen, W. M. A.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1993, 636, 3. (24) Cai, J.; Henion, J. J. Chromatogr., A 1995, 703, 667. (25) Smith, R. D.; Wahl, J. H.; Goodlett, D. R.; Hofstadler, S. A. Anal. Chem. 1993, 65, 574A. (26) Covey, T. In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, P. A., Ed.; ACS Symposium Series 619; American Chemical Society: Washington DC, 1995; pp 21-59. (27) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675. (28) Fenn, J. B.;Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64.

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Figure 3. Diagram of a structure, with intersection I1 and three channels, C1, C2, and C3, connecting the respective ports, P1, P2, and P3, to illustrate electroosmotically induced pumping principles.

the channel axis,

E(z) ) -

F dV )I dz A(z)

(4)

where V is the electric potential (assumed to be decreasing with increasing z). Inserting (1) and (4) into (2) gives, Figure 2. Photomicrographs of (a, top) a water droplet (volume ∼12 nL) forced through a channel opening by positive pressure and (b, bottom) the Taylor cone and electrospray generated at the opening applying a 3 kV potential between the microchip and a target electrode spaced a few millimeters from the channel opening.

greater than the Stern layer thickness. When an electric potential is applied across a channel of complex geometry and axially varying cross-sectional area, the current and electroosmotically induced volumetric flow rate are constant along the channel axis, assuming homogenous interfacial and fluid conditions. This implies that there will be no pressure gradients induced by electroosmotic pumping under homogenous conditions. This can be confirmed from the following equations. The flow rate, F, is given by

(2)

F ) vA(z)

where A(z) is the cross-sectional area of the flow channel as a function of the axial distance z. Ohm’s law for the channel can be written as,

∫

V ) IR(z) ) IF

L

0

dz A(z)

(3)

where I is the current, R the resistance, L the channel length, and F the resistivity of the buffer solution. The electric field strength, E, is given by the gradient of the electric potential along 1176

Analytical Chemistry, Vol. 69, No. 6, March 15, 1997

F ) I(oζF/4πη)

(5)

Since electric current is a constant along the channel, the electroosmotically induced flow rate is independent of channel cross section, assuming that the channel interface and fluid are homogeneous; i.e., the material constants , ζ, F, and η are constant. Changes in channel cross section result in modifications of the electric field strength and thus fluid velocity to maintain constant flow rate. For an interconnected channel structure, the electroosmotic flow will follow the current path as that path is the direction of the potential gradient or electric field. The above analysis indicates for a structure such as shown in Figure 3 that when an electric potential is applied between ports 1 and 2, no flow will pass from port 1 to port 3. The channels in Figure 3 are indicated by Ci, where i is the label defining the port, and the intersection is labeled as I1. For microchip channel structures considered here, the Reynolds numbers are much less than unity. Thus the viscous forces far exceed the inertial forces and there is no significant pressure generated at the intersection by the momentum of the fluid flow in channel 1. The above equations assume that the electroosmotic pumping is everywhere equivalent. Reduction of the electroosmotic fluid flow in C2 relative to C1, however, will generate an excess pressure in channel 1, allowing pumping of fluid from port 1 to ports 2 and 3. A number of methods may be used to reduce the electroosmotic flow in C2 relative to C1 including reducing the dimensions below that of the Stern layer, reducing the ζ, or increasing the surface viscosity, within channel C2. Assuming that the electroosmotic

flow velocity has been reduced to zero in this channel, then the pressure generated at the intersection I1 can be calculated using eq 1 and the equation for pressure-induced flow in channels.29 At neutral pH and field strengths of ∼350 V/cm as typically used in these experiments, the linear velocity of the electroosmotic flow is ∼2.5 mm/s, which translates to a flow rate of 1.5 nL/s, given channels of the dimensions discussed here. Approximately 95% of this flow exits the channel for the microchip described in Figure 1. Flow rates at these levels are sufficient to sustain a stable electrospray.30-33 These pumping principles were demonstrated using a microchip of the design illustrated in Figure 1. A fluorescence image of the side-arm channel intersection with homogeneous channel conditions, i.e., all channel walls have native glass surfaces and uniform ζ, is shown in Figure 4a. A positive potential relative to the side-arm reservoir was applied to the uppermost reservoir connected to the central separation channel. At the intersection, a small amount of fluid can be seen to extend past the intersection, but the bulk flow is toward the side-arm reservoir. There is no flow of fluid below the intersection. The spatial distribution of fluorescence is stationary with time. The narrow bright line seen in the image that extends below the intersection is an artifact from specularly reflected light from a channel facet. Figure 4b shows a fluorescence image taken of the same microchip intersection under the same experimental conditions except that the side-arm channel and the channel between the intersection and channel opening have been surface modified with linear polyacrylamide. The linear polyacrylamide at the walls greatly increases the surface viscosity and thus reduces the electroosmotic flow, as indicated by eqs 1 and 2. It is clear in Figure 4b that the dye solution propagates below the intersection indicating the induced pressure generated at the intersection by the spatially inhomogeneous surface viscosity. The images in Figure 4 were taken using Rhodamine B (100 µM in water), a zwitterionic dye that is near neutral charge at neutral pH. Similar images were obtained using the cationic dye Rhodamine 6G. The splitting at the side-arm intersection for ions must also include electrophoretic forces, features of which are presently under study. This electroosmotically driven fluid flow has been used to supply the fluid for electrospray from the microchip. Figure 5 shows a photomicrograph of electrospray generated from a 60% water/40% methanol solution. A voltage of 6 kV was applied to the uppermost reservoir and 4 kV was applied to the side-arm reservoir, providing ∼1.2 kV potential drop for electroosmotic pumping within the uncoated channel segment and ∼4.8 kV potential for electrospray formation. The microchip was positioned 3-5 mm from the target plate which was held at ground potential. Alternatively, of course, the side arm may be held at ground potential and the counter electrode at the elevated voltages necessary to establish the electrospray. The target is evident on right-hand side of the photograph. As shown in Figure 2b with the pressure-induced flow, Taylor cone formation is clearly evident with an emanating electrospray stream. An argon ion laser was used to illuminate the electrospray in the image shown in Figure (29) Batchelor, G. K. An Introduction to Fluid Mechanics; University Press: New York, 1967. (30) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1. (31) Wahl, J. H.; Goodlett, D. R.; Udeseth, H. R.; Smith, R. D. Anal. Chem. 1992, 64, 3194. (32) Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science 1996, 273, 1199. (33) Ramsey, R. S.; McLuckey, S. A. J. Microcolumn Sep. 1995, 7, 461.

Figure 4. CCD images of 100 µM Rhodamine B in water obtained at the intersection of a side-arm channel with a main channel on a microchip, applying a positive potential at the top channel relative to the side arm and using (a) all native glass surfaces and (b) a linear polyacrylamide surface coated side-arm channel.

5. Supplemental information on the electrospray may also be viewed by accessing a video clip. Features that should be noted are the narrowly focused plume and the stability of the spray. A mass spectrum obtained from a 10 µM solution of tetrabutylammonium iodide (molecular weight 369) electrosprayed from the microchip using electroosmotic pumping to deliver the analyte is shown in Figure 6. Voltage conditions on the microchip were the same as described above, and the sample in 60% water/40% methanol was continuously electrophoresed from the main channel to the exit where it was electrosprayed. The spectrum was averaged over 2 s to simulate peak widths that may be obtained by microchip electrophoresis at low field strengths. Assuming a flow rate of ∼1.5 nL/s, only 30 fmol of sample was consumed during this acquisition period. The tetrabutylammonium ion (M - I)+ at mass/charge 242 is clearly evident as are some solvent Analytical Chemistry, Vol. 69, No. 6, March 15, 1997

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Figure 5. Photomicrograph of the Taylor cone and electrospray generated from a 60% water/40% methanol solution that was electroosmotically pumped using a coated side-arm microchip.

Figure 6. Mass spectrum obtained from electroosmotically pumped 10 µM tetrabutylammonium iodide (in 60% water/40% methanol), averaging the data over a 2 s period and consuming ∼30 fmol of analyte. Signal intensity is shown in the upper right-hand corner.

cluster ions. Other major ions, which are not identified, may be due to fragmentation of the analyte. A heated interface, which would promote solvent evaporation for the electrospray droplets, would be expected to provide more intense (M - I)+ ions. The variation in total ion counts scanning over an m/z range from 50 to 650 for a 4.5 min interval was low (5.9% RSD for a total of 405 scans), indicating the overall stability of the electrospray source. CONCLUSIONS The microchip electrospray interface developed is simple in that it does not require any major modification of the microchip itself such as the attachment or machining of a tip at the channel exit. Electrical connections to generate the ES and drive an electrophoretic separation in microchip electrophoresis applications are made via solutions that contact the primary channel from

which the analyte is sprayed. This approach is less involved than applying a conductive coating to the exit end to establish electrical contact, a strategy employed in sheathless CE-MS interfaces.24,25,31-34 As such, the interface is not dependent upon the longevity or durability of such a coating, factors that have been a consideration in the sheathless interfaces.34 The linear polyacrylamide coating used to modify the surface of the side channel to pump solutions to the exit is covalently bound to the glass surface and is expected to be relatively stable when buffers at moderate pH are used.35 Other treatments that eliminate or reduce electroosmotic flow that may be more inert under acidic or basic conditions would, of course, be appropriate for surface modification as well. We were also able to electrospray solutions from openings that were irregular in shape as a result of polishing the electrospray end as well as from more uniform trapezoidal geometries of the channels themselves, indicating that the exact profile at these micrometer-scale dimensions are not critical. This may be because the liquid droplet as it emerges from the channel forms the tip itself from which the electrospray is established. Methods for spatially confining the droplet prior to the onset of electrospray may lower the onset potential. No particular differences where noted, however, when the exit end of the microchip was silanized to increase hydrophobicity. To preserve column efficiency as the fluid profile moves from electroosmotic flow in the primary channel to laminar flow below the side-arm intersection, the distance between the side-arm channel and electrospray exit should be minimized. This can be easily accomplished by breaking the microchip in the proper location or machining the side channel close to the exit. Overall, the interface has been shown to provide a stable electrospray and is simple and adaptable in design. For example, we are presently examining a microchip format that provides bulk fluid for electrospray when coated separation channels that have insufficient electroosmotic flow to sustain an electrospray are used. With the demonstration that microchips can be easily interfaced with mass spectrometry, the advantages afforded by the devices, including the ease of manipulating small sample volumes, the ease of integrating complex chemical reactions and separations, the high degree of automation and precision, and the potential for rapid screening resulting from construction of massively parallel analytical systems, become more readily available for MS applications. ACKNOWLEDGMENT This research was sponsored by the Department of Energy Office of Research Development. Oak Ridge National Laboratory is managed by Lockheed Martin Energy Research Corp. for the U.S. Department of Energy under Contract DE-AC05-96OR22464. Technical discussions and assistance by S. C. Jacobson and S. A. McLuckey are greatfully acknowledged. Received for review December 16, 1996.X

October

17,

1996.

Accepted

AC9610671 (34) Kriger, M. S.; Cook, K. D.; Ramsey, R. S. Anal. Chem. 1995, 67, 385. (35) Huang, M.; Vorkink, W. P.; Lee, M. L. J. Microcolumn Sep. 1992, 4, 233.

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X

Abstract published in Advance ACS Abstracts, February 15, 1997.