Anal. Chem. 2003, 75, 5697-5702
Capillary Electrophoresis Coupled On-Line with Ultraviolet Resonance Raman Spectroscopy Reyer J. Dijkstra,† Evtim V. Efremov, Freek Ariese, Udo A. Th. Brinkman, and Cees Gooijer*
Department of Analytical Chemistry and Applied Spectroscopy, Laser Centre, Vrije Universiteit Amsterdam, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Capillary electrophoresis (CE) and resonance Raman spectroscopy (RRS) with excitation in the deep ultraviolet (UV) region (λex: 244 or 257 nm) were coupled on-line. The potential of this hyphenated technique, denoted as CE-UV-RRS, for analyte confirmation/identification purposes was explored with aromatic sulfonic acids and nucleotides as test compounds. Good-quality UV-RRS spectra could be recorded on-the-fly. Identification limits for the nucleotides were in the 10-125 µg/mL range. The RRS spectra showed sufficient characteristic features to enable analyte confirmation. In addition, the identification power of UV-RRS was studied with substituted pyrenes as model compounds. The compounds were distinguishable on the basis of their RRS spectra at 244 nm. Capillary electrophoresis (CE), a powerful analytical technique with high separation efficiency, is usually combined with a UV absorption detector. However, for analyte identification purposes, it is necessary to couple CE with spectroscopic detection techniques. To date, emphasis is on the coupling with mass spectrometry (MS), which is rapidly becoming a mature technique. The possibility to couple CE with identification techniques which provide information complementary to MS, such as nuclear magnetic resonance (NMR), fluorescence line-narrowing spectroscopy (FLNS), Fourier transform infrared (FTIR) or Raman spectroscopy, (RS) would be welcome. A review covering the stateof-the-art was published a few years ago.1 Regarding the hyphenation of CE and the vibrational techniques, FLNS, FT-IR and RS, there are various problems that hamper its realization. FLNS requires freezing of the separation to 4 K and is only applicable to fluorescent compounds.2 Because of the IR absorption of both the fused-silica capillary and the aqueous solutions that are typically used in CE, the direct coupling with FTIR is not straightforward; nonetheless, both on-line3 and at-line4 combinations were recently reported. On-line CE-RS looks more promising in this respect; however, because of the lack of * Corresponding author. E-mail: Fax: +31-20-4447543.
[email protected]. † Current address: Department of Applied Physics, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. (1) Kok, S. J.; Velthorst, N. H.; Gooijer, C.; Brinkman, U. A. Th. Electrophoresis 1998, 19, 2753-76. (2) Zamzow, D.; Lin, C.-H.; Small, G. J.; Jankowiak, R. J. Chromatogr., A 1997, 781, 73-80. (3) Kolhed, M.; Hinsmann, P.; Svasek, P.; Frank, J.; Karlberg, B.; Lendl, B. Anal. Chem. 2002, 74, 3843-48. (4) Jarman, J. L.; Todebush, R. A.; de Haseth, J. A. J. Chromatogr., A 2002, 976, 19-26. 10.1021/ac034555z CCC: $25.00 Published on Web 09/12/2003
© 2003 American Chemical Society
sensitivity of RS, analyte detectability is rather poor. Hyphenation of CE and RS is only viable if high laser powers can be applied and suitable on-line preconcentration techniques, such as isotachophoresis5-7 or field-amplified injection,8,9 can be utilized. The general approach is to preconcentrate the analytes so that at the detection window their concentration is higher than 10 mM. Under such conditions, good-quality conventional Raman spectra for ribonucleotides have been recorded with initial concentrations down to 5 µM.5 It is well-known that much higher signals can be obtained when using special modes of RS, that is, in resonance Raman (RRS) or surface-enhanced Raman (SERS) spectroscopy. For SERS, the analyte has to be adsorbed onto a suitable metallic substrate, for instance, a silver or gold colloid or a roughened metal surface. Recently, SERS was coupled to CE in the at-line mode.10-12 Online coupling, which is rather difficult, since colloids have to be introduced in the CE system, has also been reported.13 For RRS, there are no special requirements that complicate the on-line coupling; the only condition is that the laser excitation wavelength has to be tuned within an electronic absorption band of the analyte. In this way, a 102-103-fold enhancement should be expected, and even factors up to 108 have been reported.14 The problems to overcome in CE-RRS are the fluorescence background that usually overwhelms the Raman signals and, additionally, the lack of available laser wavelengths. Until now, in the literature, the focus of attention has been on RRS in the visible region. In fact, CE-RRS was realized more than a decade ago. Two dyes, methyl red and methyl orange, were excited at 532 nm, and detection limits as favorable as 10-7 M were obtained.15 However, few compounds show absorption in the visible region; most require UV excitation, which implies that (5) Walker, P. A.; Kowalchyk, W. K.; Morris, M. D. Anal. Chem. 1995, 67, 4255-60. (6) Walker, P. A.; Morris, M. D.; Burns, M. A.; Johnson, B. N. Anal. Chem. 1998, 70, 3766-69. (7) Walker, P. A.; Morris, M. D. J. Chromatogr., A 1998, 805, 269-75. (8) Kowalchyk, W. K.; Walker, P. A.; Morris, M. D. Appl. Spectrosc. 1995, 49, 1183-88. (9) Walker, P. A.; Shaver, J. M.; Morris, M. D. Appl. Spectrosc. 1997, 51, 139499. (10) DeVault, G. L.; Sepaniak, M. J. Electrophoresis 2001, 22, 2303-11. (11) He, L.; Natan, M. J.; Keating, C. D. Anal. Chem. 2000, 72, 5348-55. (12) Seifar, R. M.; Dijkstra, R. J.; Gerssen, A.; Ariese, F.; Brinkman, U. A. Th.; Gooijer, C. J. Sep. Sci. 2002, 25, 814-18. (13) Nirode, W. F.; DeVault, G. L.; Sepaniak, M. J. Anal. Chem. 2000, 72, 186671. (14) Asher, S. A. Anal. Chem. 1993, 65, 59A-66A. (15) Chen, C. Y.; Morris, M. D. J. Chromatogr. 1991, 540, 355-63.
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CE-UV-RRS will be more broadly applicable. In addition, in the deep-UV region (
