Anal. Chem. 1997, 69, 2537-2540
Pulsed UV Laser-Induced Stationary Capillary Vibration for Highly Sensitive and Direct Detection of Capillary Electrophoresis Tamao Odake, Takehiko Kitamori, and Tsuguo Sawada*
Department of Applied Chemistry, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113, Japan
A stationary wave of the capillary vibration effect was successfully induced by a series of short laser pulses. This wave could be applied to highly sensitive detection of capillary electrophoresis as well as the already reported capillary vibration induced by an intensity-modulated CW laser (CVL effect). Generally, pulses with much shorter width than the period of the natural frequency of the vibrating system cannot induce a standing vibration. However, utilizing the time constant of CVL determined by heat dissipation time, we found conditions which could induce a stable stationary wave of the capillary by a series of nanosecond light pulses. We used the KrF excimer laser operated at 248 nm with a pulse width of 60 ns and output of ∼10 µJ/pulse as the CVL excitation source and applied it to highly sensitive detection of nonderivatized amino acids at the femtomole level. The sensitivity was at least 2 orders of magnitude superior to that of a commercially available UV absorbance detector. This technique extends the CVL’s spectral regions. For example, in the UV region, where many biological materials have significant absorption bands, this technique will extend analytical applications in capillary electrophoresis by eliminating the need for a derivatization process. Over a decade has passed since capillary electrophoresis (CE) was first proposed as a high-performance separation technique.1 Its advantage of requiring a sample quantity of less than a nanoliter has been utilized, with promising results being demonstrated for single-cell analysis,2,3 zeptomole detection,4 and on-column reactions.5,6 In such microscale analyses, electrophoretically separated sample components cannot be detected without a sensitive detector. Laser-induced fluorometry (LIF), mass spectrometry (MS), and electrochemical detection (ECD) have all been studied for sensitive detection of CE. To maintain a superior separation ability, on-column detection is desirable for ultramicroanalysis. However, the latter two detection methods, MS and ECD, have the disadvantage that they are off- or end-column detections, or contact detections. Even LIF detection is not perfect, because only highly fluorescent substances can be detected with high sensitivity and fluorescent derivatization of the analyte is usually * To whom all correspondence should be addressed. Fax: +81-3-3815-6543. E-mail:
[email protected]. (1) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (2) Hogan, B. L.; Yeung, E. S. Anal. Chem. 1992, 64, 2841-2845. (3) Xue, Q.; Yeung, E. S. Anal. Chem. 1994, 66, 1175-1178. (4) Chen, T. K.; Luo, G.; Ewing, A. G. Anal. Chem. 1994, 66, 3031-3035. (5) Avila, L. Z.; Whitesides, G. M. J. Org. Chem. 1993, 58, 5508-5512. (6) Chang, H.-T.; Yeung, E. S. Anal. Chem. 1993, 65, 2947-2951. S0003-2700(96)01135-3 CCC: $14.00
© 1997 American Chemical Society
needed, which determines the lower limit of sample quantity and applicable cases. To solve the above problems, we and other groups have studied and developed a photothermal detection technique for sensitive detection of CE.7-14 Compared with other detection techniques, photothermal detection has a number of advantages. First, the sensitivity is high. The lower detection limit is 10-7 in absorbance, which is 3 orders of magnitude superior to that of a conventional absorbance detection technique and is no worse than that of the LIF method. Second, any analyte that can convert laser radiation to heat can be detected by this technique. The selectivity and applicability are similar to those of a conventional absorbance detection method. Hence, the photothermal methods allow direct detection of analyte without any derivatization or labeling. Third, the detection scheme provides on-column detection. We have earlier observed the capillary vibration induced by laser (CVL) effect,15 which is a kind of photothermal effect, and proposed CVL detection as a highly sensitive on-column technique for CE.10,12,14 Because smaller diameter capillaries have a larger CVL amplitude, CVL detection is most effective with rather small diameter capillaries and, as such, is expected to be suitable for ultramicroscale detection. Compared with another means of photothermal detection, thermooptical detection,7-9,11 CVL detection is advantageous in that it is not easily affected by optical properties of the liquid flowing through the capillary. Previously, a modulated continuous wave (CW) laser was used as the CVL excitation source. When a capillary is irradiated by intensitymodulated CW laser light, heat is generated photothermally to cause tension fluctuation, which in turn causes capillary vibration. Because the capillary vibration is a forced vibration, it can be induced at any frequency of the same order as or lower than the natural frequency (typically around 1 kHz), and we can choose the modulation frequency that gives the best signal-to-noise (S/ N) ratio. The amplitude of the capillary is inversely proportional to modulation frequency. However, the S/N ratio is not good at lower modulation frequencies (
