Anal. Chem. 1998, 70, 3766-3769
Isotachophoretic Separations on a Microchip. Normal Raman Spectroscopy Detection Patrick A. Walker, III† and Michael D. Morris*
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055 Mark A. Burns and Brian N. Johnson
Department of Chemical Engineering, The University of Michigan, Ann Arbor, Michigan 48109-2136
Isotachophoretic separations of the herbicides paraquat and diquat are performed in a glass microchip etched channel and monitored on-chip by normal Raman spectroscopy. The 40-µm-wide and 75-µm-deep separation channels are chemically etched in a serpentine design to 21-cm total length. A 120-µm-thick glass cover slip is used to seal the channels. Separation field strengths up to 380 V/cm are used. The microchip is directly coupled to a Raman microprobe. No interfacing is required. Raman spectra are generated with a 2-W, 532-nm NdYVO4 laser and collected at 8-cm-1 resolution with a holographic transmissive spectrograph and a cryogenically cooled CCD. Data acquisition is at 2-5 spectra/s. Raman isotachopherograms of the pesticides at starting concentrations as low as 2.3 × 10-7 M (60 ppb paraquat/ 80 ppb diquat) are presented. There is intense current interest in chemical manipulations and separations in microfabricated systems. Many familiar analytical separation techniques have been demonstrated in the microchip format. These include liquid chromatography,1 micellar electrokinetic chromatography,2 electrochromatography,3 and electrophoresis.4,5 Electrically driven separations appear be the well suited for miniaturization because they do not require either high pressures or miniaturized mechanical pumping systems. Microchips have large surface areas compared to capillaries with similar internal cross sections. The chips efficiently dissipate heat generated by current flow. Even without active cooling, operation of electrically driven separations at high fields proceeds with minimal sample heating. Heat dissipation is particularly useful for isotachophoresis (ITP) because high-concentration leading and trailing electrolytes are used. High voltage reduces running times and therefore decreases interdiffusion of analyte zones which limit resolution.
Fluorescence6 and UV7 spectroscopies are the most common detection techniques for microchip separations. These spectroscopies are excellent for quantification but contain limited qualitative information. Electrospray ionization from a microchip into a mass spectrometer has been demonstrated, although not integrated with a separation step8,9 We have previously demonstrated that normal Raman spectroscopy can be used as an on-line detector principle for capillary electrophoretic separations.10-14 A major advantage is that little or no interfacing is required. With conventional capillaries, Raman microprobes can be employed with no changes other than that required for high-voltage isolation. Optical fiber probes can be used to perform on-capillary Raman spectroscopy inside a conventional safety chamber. Because water has a weak, but real Raman spectrum, preconcentration is needed to achieve useful detection limits. ITP has proven especially useful. With tightly focused laser light and acquisition of 1-2 spectra/s, ITP/Raman spectroscopy can be used with starting concentrations below 10-7 M. In this paper, we demonstrate on-chip Raman detection of ppb levels of the herbicides paraquat and diquat separated by isotachophoresis. We show that on-chip Raman spectroscopy can be performed on an unmodified Raman microprobe at 2-5 spectra/ s. EXPERIMENTAL SECTION Microchip Fabrication. Photolithographic microfabrication of microchips was performed as described elsewhere.15 Briefly,
† Present address: BetzDearborn Corp., 4636 Somerton Rd., Trevose, PA 19053-6783. (1) Manz, A.; Miyhara, Y.; Miura, J.; Watanabe, Y.; Miyagi, H.; Sato. K. Sens. Actuators 1990, B1, 249. (2) Moore, A. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1995, 67, 4184. (3) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 2369. (4) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 2637. (5) Harrison, D. J.; Manz, A.; Fan. Z.; Ludi, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926.
(6) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114. (7) Van der Moolen, J. N.; Poppe, H.; Smit, H. C. Anal. Chem. 1997, 69, 4220. (8) Xue, Q.; Foret, F.; Dunayaeskiy, Y. M.; Zavrachy, P. M.; McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426. (9) Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 1997, 69, 1174. (10) Kowalchyk, W.; Walker, P. A., III; Morris, M. D. Appl. Spectrosc. 1995, 49, 1183. (11) Walker, P. A., III; Kowalchyk, W.; Morris, M. D. Anal. Chem. 1995, 67, 4255. (12) Li, H.; Walker, P. A., III; Morris, M. D. J. Microcolumn Sep., in press. (13) Walker, P. A., III; Morris, M. D. J. Chromatogr. 1998, 805, 269. (14) Walker, P. A., III; Shaver, J. M.; Morris, M. D. Appl. Spectrosc. 1997, 51, 1394. (15) Burns, M. A.; Mastrangelo, C. H.; Sammarco, T. S.; Man, F. P.; Webster, J. R.; Johnson, B. N.; Forester, B.; Jones, D.; Fields, Y.; Kaiser, A. R.; Burke, D. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5556-5561.
3766 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
S0003-2700(98)00195-4 CCC: $15.00
© 1998 American Chemical Society Published on Web 08/08/1998
Figure 1. Diagram of Raman spectroscopy/microchip isotachophoresis system. Component details in text.
Figure 2. Raman spectra of paraquat and diquat taken with the Raman microprobe, 500-mW, 532-nm excitation, 1-s integration.
a 21-cm serpentine channel geometry was chemically etched into a glass microscope slide. The channel was etched to approximately 40 µm wide and 75 µm deep using a 1:1 mixture of HF/H2O. Since the angles of the side walls are a little less than 45°, the width at the bottom is ∼75 µm, while the width at the top (near the cover slip) is about 175-200 µm. However, the exoxy cement used to bond the cover slip fills the edges of the channel, reducing the width is ∼175 µm. Access points to the channel were electrochemically drilled into the cover slip prior to bonding.16 To drill holes in the channel, 37 V was applied to a metal point touching the glass surface in a 50 wt % sodium hydroxide solution. The point was slowly pushed through the glass (∼5-10 s). Reservoirs were constructed around cover slip holes from polyethylene pipet tips and fastened to the cover slip with epoxy cement. A 120-µm-thick cover slip was sealed over the channel with UV-cure epoxy cement. The glass cover slip was placed on top of the silicon substrate, and optical adhesive (SK-9 Lens Bond, Sumers Laboratories, Fort Washington, PA) was applied to the edge of the channel using a small paintbrush and allowed to wick between the substrate and cover slip. The cement was cured under a UV lamp for 24 h. The thickness of the layer was tested with optical and Raman microscopy and found too low to measure (
