Anal. Chem. 2001, 73, 1080-1083
Sequential Electrospray Analysis Using Sharp-Tip Channels Fabricated on a Plastic Chip Cheng-Hui Yuan and Jentaie Shiea*
Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, Taiwan
A disposible plastic (poly(methyl methacrylate)) chip containing eight open channels (375 µm in width, 300 µm in depth, 1.25 cm in length) capable of performing sequential electrospray analysis is described. The channels were constructed by simply cutting the plastic chip with a sharp knife. One end of each channel was fabricated to a sharp end, while the other was connected to a sample well. The high voltage required for electrospray was introduced into the sample solution (70% methanol/ water) via a copper electrode inserted in the sample well. The solution in the sample well was continuously drawn to the sharp end of the channel by capillary action and by applying the electrospray voltage at the sample reservoir. A stable and fine Taylor cone was observed at the exit of the channel. Chemical modification on the surface of the channel was not required to decrease wettability. The octagonal plastic chip allowed sequential electrospray analyses of eight samples. The technique was also shown to be useful in rapidly determining active ingredient in a commercial tablet.
Generating electrospray (ES) directly from glass microchips has recently received considerable attention because of the easy construction of the microchips, short time required for analysis, small sample usage, and potential to be incorporated into capillary electrophoresis.1-3 Directly generating electrospray from the planar edge of a microchannel on the glass chip is rather difficult because the fluid in the microchannel tends to spread over the surface before the onset of electrospray.3 This diffusion increases the electrospray threshold voltage and reduces the sensitivity and efficiency of separations performed on the device. Two methods have been developed to solve the problem: (1) modification of the glass microchannel surface to decrease the wettability, especially at the microchannel exit,1-3 and (2) attachment of a nanospray tip to the microchannel exit.4,5 Although both methods have yielded promising experimental results, extensive work is (1) Xue, Q. F.; Foret, F.; Duanyevskiy, Y. M.; Zavracky, P. M.; Mcgruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426. (2) Xue, Q. F.; Duanyevskiy, Y. M.; Foret, F.; Karger, B. L. Rapid Commun. Mass Spectrom. 1997, 11, 1253. (3) Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 1997, 69, 1174. (4) Lazar, I. M.; Ramsey, R. S.; Sundberg, S.; Ramsey, J. M. Anal. Chem. 1999, 71, 3627. (5) Bings, N. H.; Wang, C.; Skinner, C. D.; Colyer, C. L.; Thibault, P.; Harrison, D. J. Anal. Chem. 1999, 71, 3292.
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required to fabricate and manufacture the chip. This study designs a simple and inexpensive channel capable of generating an electrospray which could be easily constructed in an ordinary laboratory. Polymer substrates such as polyethylene (PE), polypropylene (PP), poly(vinyl chloride) (PVC), poly(tetrafluoroethylene) (PTFE), ethylene/vinyl acelate (EVA), poly(butylene terephthalate) (PBTP), and poly(methyl methacrylate) (PMMA) have been shown to be promising alternatives for producing electroosmotic flow in capillary electrophoresis.6-11 These synthetic polymer-based materials used in a microfluidic system have the advantage of being more easily manipulated than the silica-based substrates. Moreover, the inherent hydrophobic nature of most synthetic polymers allows the channel to be applied in the analysis of biological compounds without modifying the surface to reduce wall surface adsorption of the analyte.9-11 PMMA is generally known to be the least hydrophobic of the common plastic materials. Electrophoresis based on PMMA capillary tubing and a microchip has been investigated, and the results indicate that an electroosmotic flow could be generated in such a capillary.9-11 Accordingly, this work develops a device capable of generating a stable electrospray from the opening channel manipulated on a PMMA chip. Sequential analyses of the standards are conducted using a PMMA chip containing eight channels. Rapid determination of the active ingredient in a commercial tablet by this device is also demonstrated. EXPERIMENTAL SECTION Figure 1 schematically depicts the open channel designs employed in the experiments. The channels can be easily fabricated on the PMMA chip as it is not as hard or fragile as a glass chip. Indeed, a square PMMA chip was fabricated into an octagon using a saw. Each arm of the chip was then filed to a sharp end (Figure 1). Both sides of the sharp end were polished with an abrasive sheet (1000 grit). The chip was then placed on a milling machine and the channel was constructed by cutting through the center of each arm with a sharp knife. Eight open channels were constructed on the octagonal chip. The diameter of the channel depended on the size of the knife employed. (6) Schutzner, W.; Kenndler, E. Anal. Chem. 1992, 64, 1991. (7) Chen, Y. H.; Wang, W. C.; Young, K. C.; Chang, T. T.; Chen, S. H. Clin. Chem. 1999, 45, 1938. (8) Locascio, L. E.; Perso, C. E.; Lee, C. S. J. Chromatogr., A 1999, 857, 275. (9) Bayer, H.; Engelhardt, H. J. Microcolumn Sep. 1996, 8, 479. (10) Lee, M. L.; Chen, S. J. Microcolumn Sep. 1997, 9, 57. (11) Chen, Y. U.; Chen, S. H. Electrophroresis 2000, 21, 165. 10.1021/ac001305z CCC: $20.00
© 2001 American Chemical Society Published on Web 02/14/2001
Figure 2. Photograph of the electrospray at the tip of the channel cut on a PMMA chip.
chip was rotated to the next sample well. All chemicals used in this study were purchased from Sigma or Aldrich and used without further purification.
Figure 1. Schematic diagram of the PMMA chip electrospray arrays used to analyze sequentially eight samples (A, copper electrode; B, acrylic bar; C, electrical cable; D, plastic box; E, spring on an acrylic rod).
Channels with width 375 µm ((25 µm), depth 300 µm ((20 µm), and length 1.25 cm were constructed. A sample reservoir (3 mm in diameter and 0.5 mm in depth) was constructed by drilling a well at the end of each channel. The chip was set on a rotary acrylic box mounted on an XYZ translation stage. One microliter of the sample solution was deposited in the sample well and the chip was set in front of the mass spectrometer with the channel tip ∼5 mm away from the sampling skimmer of an interface plate on an PE Sciex API 1 mass spectrometer. The position of the channel tip relative to the sampling skimmer on the interface of the mass spectrometer was adjusted by the translation stage. The quadrupole mass analyzer was scanned from m/z 10 to 2000 at a rate of ∼1.5 s/scan. The electrospray interface chamber was maintained at 55 ( 1 °C. The high voltage (3800 V) required for electrospray was conducted through the sample solution by manually immersing a copper electrode into the sample well.12-14 The electrode was connected to a high-voltage power supply (EH10R10) through an electrical cable immersed in the groove cut on the acrylic bar (see Figure 1). The electrode was held on an acrylic bar that can be manually moved up and down to perform sequential analyses. As the sample well was manually rotated underneath the electrode, the electrode was put into the well and the high-voltage power supply was switched on. After the analysis was completed, the power supply was turned off, the electrode was raised, and the (12) Shiea, J. T.; Wang, C. H. J. Mass Spectrom. 1996, 32, 247. (13) Kuo, C. P.; Shiea, J. Anal. Chem. 1999, 71, 4413. (14) Lee, C. Y.; Shiea, J. Anal. Chem. 1998, 70, 2757.
RESULTS AND DISCUSSION Construction of a microchannel on a glass chip conventionally requires thoroughly cleaning of the chip surface, metal deposition, and photolithographic patterning.15-18 The surface of the microchannel must also be chemically treated because wetness on the edge of the surface prevents the formation of a well-focused electric field essential for generating a stable electrospray.1,3 Additionally, spreading the solution at the microchannel exit can potentially lead to cross-contamination of the microchip with a multichannel configured in parallel. The channel manipulated on a PMMA chip has a hydrophobic surface so modification to reduce the wettability of the surface of the channel is not required. In this study, an open channel (375 µm in width and 300 µm in depth) was simply constructed by cutting the plate with a sharp knief on a computer-controlled milling machine. Observation with a microscope showed that the sample solution containing more than 70% methanol was immediately delivered from the sample well to the tip of the channel by capillary action as soon as the sample solution was deposited in the sample well. However, as the high voltage (3800 V) required for electrospray was added into the solution contained in the open channel with a flat edge, the fluid tended either to accumulate at the channel exit or to spread over the channel prior to the onset of electrospray. When the exit of the channel was sharpened (see Figure 1), no such phenomenon was observed and a stable electrospray was generated directly from the center of the channel. Figure 2 displays a low-magnification photograph of a Taylor cone and the electrospray generated from the methanol solution by this device. The sharp exit confined the fluid emanating from the channel, helping to maintain a parabolic pattern so the electric field could be focused at the tip of the flow. A stable and fine electrospray was immediately induced from the tip of the parabolic flow. (15) Jacobson, S. C.; Culbertson, C. T.; Daler, J. E.; Ramsey, J. M. Anal. Chem. 1998, 70, 3476. (16) Koutny, L. B.; Schmalzing, D.; Taylor, T. A.; Fuchs, M. Anal. Chem. 1996, 68, 18. (17) Woolley, A. T.; Sensabaugh, G. F.; Mathies, R. A. Anal. Chem. 1997, 69, 2181. (18) Zhang, B.; Liu, H.; Karger, B. L.; Foret, F. Anal. Chem. 1999, 71, 3258.
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Figure 3. Positive electrospray mass spectra of (a) myoglobin, (b) insulin, (c) cytochrome c, and (d) bradykinin obtained from the channels cut on an octagonal PMMA chip.
The fluid in the sample well was continuously drawn through the channel without electroosmotically induced pressure or any external pressure as the electrospray was initiated at the tip of the channel. The sample consumption was approximately 0.5-1 µL/min. The solution containing less than 70% methanol was too viscous to flow through the channel. In this case, external pressure must be applied to the solution in the sample well. Several sources of external pressure have been described, including a microchannel with a syringe pump, an electroosmotic pump, a pressurized inlet reservoir, and a micromachined pneumatic nebulizer.1,3,18 Reducing the diameter of the channel to increase the capillary action may also help to deliver a solution containing more water. A chip with multiple channels was used to analyze sequentially eight samples. The PMMA chip was fabricated in an octagonal configuration (Figure 1). This configuration prevents crosscontamination of the solution due to spreading at the exit of the channels. Each sample solution (10-6 M and 1 µL) was deposited into two consecutive sample wells. Figure 3 displays the ES mass spectra for four different standardssmyoglobin, cytochrome c, insulin, and bradykinin. As it can be seen, the ES mass spectra were nearly the same as those obtained by the electrospray using a capillary. It was also found that both positive and negative mass spectra obtained from the same sample solution deposited in different sample wells remained the same (data not shown). Channel exits with different angles (30°, 60°, 90°, and 120°) were fabricated and tested for their electrospray generation capability (Figure 4). Figure 4 displays the ES mass spectra of myoglobin obtained from these channels. The parabolic liquid flow pattern was maintained as it emanated from each of the channels. A stable electrospray was generated from each of these channels, and no significant difference was detected in the ion signal stability or the onset voltage of the electrospray. 1082 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
The open channel used in this study had a larger diameter than those used for capillary electrophoresis (CE). Although decreasing the diameter of the channel reduces the flow rate and results in lower sample consumption, a microchannel with a diameter of less than 100 µm cannot yet be repeatedly constructed by just cutting with a knife because the precision of the channel diameter dramatically decreases with the size. This may due to the malleable feature of PMMA resulting in more channel deformation when a small channel is constructed. However, the imprinting method developed by Martynova et al. demonstrated that microchannels with an average diameter of 32 µm (4% standard deviation) can be repeatedly constructed on the plastic chip.19,20 This technique is currently being adopted to further reduce the size of channels on PMMA chips in our laboratory. Another difficulty with PMMA is the large variation (10%) of electroosmotic mobility (EOF) due to adsorption of biomolecules.21 To circumvent the problem, a disposable chip is suggested for routine sample analysis. An application of using the open channel for electrospray is rapid analysis of a dirty sample with little sample pretreatment. No contaminating or clogging problems occur because a relatively wide and disposable chip is used. Figure 5 shows the electrospray mass spectrum of sildenafil (MH+, m/z 475) present in a commercial tablet.22 The sample treatment was extremely simple: a small piece of the tablet (∼5 mg) was chopped and smashed; two drops of 70% methanol solution (∼100 µl) were mixed with the smashed sample to form a slurry solution. Two (19) Martynova, L.; Locascio, L. E.; Gaitan, M.; Kramer, G. W.; Christensen, R. G.; MacCrehan, W. A. Anal. Chem. 1997, 69, 4783. (20) Xu, J.; Locascio, L.; Gaitan, M.; Lee. C. S. Anal. Chem. 2000, 72, 1930. (21) Locascio, L. E.; Perso, C. E.; Lee, C. S. J. Chromatogr., A 1999, 857, 275. (22) 2. Kuo, C. P.; Yuan, C. H.; Shiea, J. J. Am. Soc. Mass. Spectrom. 2000, 11, 464.
Figure 5. Positive electrospray mass spectrum of sildenafil (MH+, m/z 475) obtained from the channel cut on a PMMA chip.
Figure 4. Positive electrospray mass spectra of myoglobin obtained from the channels with the exit sharpened at different angles: (a) 30°, (b) 60°, (c) 90°, and (d) 120°.
microliters of the solution was deposited in the sample well via a micropipet. The mass spectrum was obtained immediately after the high-voltage supply was switched on. The sample pretreatment took less than 2 min because filtration and concentration were unnecessary. CONCLUSION This study generated an electrospray from an open channel with a sharp exit on a PMMA chip. Surface modification of the
channel to decrease the wettability was unnecessary. Manufacture of the reporting system required only simple machine fabrication work and the cost was extremely low. Accordingly, the system can be easily implemented in most chemistry and biochemistry laboratories. Mass production of the disposable device might be possible for routine sample analysis. The device is also adequate to analyze a dirty sample solution with minimal sample pretreatment. The sample solutions containing small particles (e.g., suspended fibers and particles, large cells, inorganic catalysts, or tiny glass beads) may be directly analyzed without filtration, extraction, or concentration. This will greatly shorten the time required for analysis. The technique may be useful in rapidly screening a large number of unknown samples. Additional investigations related to this technique are now undergoing in our laboratory. These include (1) automation of sample switching and electrode movement and (2) reduction of the size of the microchannel so the sample consumption rate can be decreased and on-line electrophoresis can be performed. ACKNOWLEDGMENT The authors thank the National Science Council of Taiwan for financially supporting this research. Received for review November 6, 2000. Accepted January 16, 2001. AC001305Z
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