Anal. Chem. 1QQZ.6 4 , 964-967
961
to physiological samples. Further design of the host compounds to enhance the base-pairing ability and increase the lipophilicity would afford more sensitive and selective potentiometric sensors for nucleotides.
Ammann. D.: Huser. M.: Krautler. 6.;Rusterholz, 8.; Schubeas, P.; Lindemann, E.; Halder, E.; Simon, W. Helv. Chim. Acta 1988, 69, 849-854. Chaniotakis, N. A.; Chasser, A. M.; Meyerhoff, M. E.; Groves, J. T. Anal. Chem. 7988s60. 185-188. Hodlnir, A.: Jyo, A. Chem. Len. 1888. 993-998. Hodinir, A.; JYO, A. Anal. Chem. 1889, 67, 1169-1171. Daunert, S.; Bachas, L. 0. Anal. Chem. 1989, 61, 499-503. Ohki, A.; Yamura, M.; Kumamoto, S.; Maeda, S.; Takeshlta, T.; Takagi, M. Chem. Lett. 1989, 95-98. Ohki, A.; Yamura, M.; Takagi, M.: Maeda, S. Anal. Sci. 1990, 6 , 505-588. Wotring, V. J.; Johnson, D. M.; Bachas, L. 0. Anal. Chem. 1990, 62, 1506-1510. Umezawa, Y.; Kataoka, M.; Takami, W.; Kimura, E.; Koike, T.; Nada, H. Anal. Chem. 1888, 6 0 , 2392-2396. Kataoka, M.; Naganawa, R.; Odashlma, K.; Umezawa, Y.; Klmura, E.; Koike, T. Anal. Lett. 1989. 22, 1089-1105. Naganawa, R.; Kataoka. M.; Odashima, K.; Umezawa, Y.; Klmura, E.; Koike, T. Bunsekl Kagaku 1990, 39, 871-676. Umezawa, Y.; Sugawara, M.; Kataoka, M.; Odashima, K. I n Ion-Selectlve Electrodes, 5 ; Pungor, E., Ed.; A k a h l e i Kiad6 (Pergamon Press): Budapest (Oxford), 1989; pp 21 1-234. Odashlma, K.; Umezawa, Y. I n Biosensor Technology; Buck, R. P., Hatfield, W. E.. Umak, M., Bowden, E. F., Eds.; Marcel Dekker: New York, 1990: Chapter 6. Odashima, K.; Sugawara, M.; Umezawa, Y. Trends Anal. Chem. 1990, 10, 207-215. Furuta, H.; Magda, D.; Sessler, J. L. J . Am. Chem. Soc. 1981, 773, 978-985. Furuta. H.; Furuta, K.; Sessler, J. L. J . Am. Chem. SOC. 1991, 713. 4708-4707. Recommendations for Nomenclature of Ion-Selective Electrodes. Pure Appl. Chem. 1976, 46, 129-132. Umezawa, K.; Umezawa, Y. I n CRC Handbook of Ion-Seknve Electrodes: SelecdMtyCoefticients; Umezawa, Y., Ed.; CRC Press: Boca Raton, FL, 1990; pp 3-9. Uemasu, I.; Umezawa, Y. Anal. Chem. 1982. 54, 1198-1200. Lindmr, E.; T&h, K.; Pungor, E.; Umezawa. Y. Anal. Chem. 1884, 56, 808-810. Lindner, E.: Tbth, K.; Pungor, E. Pure Appl. Chem. 1988, 56, 469-479.
ACKNOWLEDGMENT We are grateful for the collaboration made possible by the JSPS Joint Research Program, organized by Eiichi Kimura, Department of Medicinal Chemistry, Hiroshima University School of Medicine, Hiroshima, Japan, and sponsored by the Japan Society for the Promotion of Science. The macrocyclic polyamine 2 used in the present study was kindly provided by Eiichi Kimura. We gratefully acknowledge support for the present study from the Grant-in-Aids for Scientific Research from the Ministry of Education, Science and Culture to Y.U. and from the Texas Advanced Research Program to J.L.S. J.L.S. also acknowledges the National Science Foundation (P.Y.I. Award 1986), the Sloan Foundation (Fellowship 1989), and the Camille and Henry Dreyfus Foundation (TeacherScholar Award 1988). REFERENCES Koryta, J. Anal. Chlm. Acta 1980, 223,1-30. Soisky, R. L. Anal. Chem. 1990, 62, 21R-33R. Janata, J. Anal. Chem. 1990, 62,33R-44R. Colllson, M. E.; Meyerhoff, M. E. Anal. chem. 1990, 62, 425A-437A. Pungor, E.; Lindner, E.; Tbth, K. Fresenius J . Anal. Chem. 1990, 337, 503-507. Janata, J. Chem. Rev. 1980, 90, 691-703. Ammann, D.; Mod, W. E.; Anker. P.; Meier, P. C.; Pretsch, E.; Simon, W. Ion-Selective Electrode Rev. 1983, 5 , 3-92. Shono, T. Bunsekl Kagaku 1984, 33,E449-E458. Wuthler, U.; Pham, H. V.; Zund, R.; WeRi, D.;Funck, R. J. J.; Bezegh, A.; Ammann, D.; Pretsch, E.; Simon, W. Anal. Chem. 1984. 56, 535-538. SchuRhess, P.; Ammann, D.;Krautler, B.; Caderas, C.; Stepinek, R.; Simon, W. Anal. Chem. 1985, 57, 1397-1401.
RECEIVED for review August 9, 1991. Accepted January 31, i992.
Raman Spectrometry with Metal Vapor Filters R. Indralingam,+J. B. Simeonsson,* G.A. Petrucci, B. W.Smith, and J. D. Winefordner*
Department of Chemistry, University of Florida, Gainesville, Florida 3261 1-2046 INTRODUCTION Perhaps the most important instrumental consideration in the detection of Raman scattering is the rejection of specular and Rayleigh scattering of the excitation source. In conventional scanning Raman systems, this entails a sacrifice in detection efficiency through the use of large double spectrometers with relatively low optical throughput. In Fourier transform based approaches, reduction of specular and Rayleigh scattering is critical due to the so-called multiplex disadvantage inherent in the FT technique. A highly efficient filter would make possible more compact Raman instrumentation using modem multichannel detection methods such as the CCD.’ The ideal fiter for either dispersive or FT methods would attenuate very efficiently only within the spectral profile of the laser source and be highly transparent a t wavelengths removed from the exciting wavelength. This requires a narrow bandpass filter with a very steep absorption edge or a ‘notch” absorption filter with an exceptionally narrow absorption profile. In practice, neither has yet been achieved. *Author to whom reprint requests should be sent. Present Address: Department of Chemistry, Stetson University, Deland, FL 32720. Present Address: Department of the Army, U.S. Army Laboratory Command, Ballistic Research Laboratory, Aberdeen Proving Ground, MD 21005-5066.
*
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The most commonly used optical filter for Rayleigh scatter is the double or triple monochromator which is a part of routine Raman i n s t r u m e n t a t i ~ n . ~Since ~ ~ a broad, tailed Rayleigh scatter peak is observed even with these spectrometers, other filters have been developed for use in both dispersive and FT-Raman spectrometry. An iodine vapor cell has been used with an argon ion laser source and a single monochromator.*v5 Molecular iodine has an absorption band a t the argon ion laser wavelength of 514.5 nm and has been shown to remove elastically scattered light in surface-enhanced Raman spectroscopy6 as well as spontaneous Raman spectrometry. A filter spectrograph’ combines a line rejection filter with a dispersive spectrograph. The instrument is a modified double monochromator having a cylindrical mirror positioned so that the laser line will exit through an aperture in the mirror after preliminary dispersion of the scattered radiation. All other spectral lines are reflected and reformed into an output light signal containing all of the Raman spectral lines except the rejected laser line. The dispersion spectrograph is coupled to the line rejection filter and produces the Raman spectrum from the output light signal. A variation of this instrument is the variable band pass filter.* Ruby laser radiation is absorbed in an unpumped ruby crystal, and an array of ruby crystal cubes has been used as 0 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992 965 a rejection filter against ruby laser light in lidar scattering e~perimenta.~ Since the absorption of the ruby crystal depends on the polarization of the radiation, overall attenuation is low with incompletely polarized incident light; alignment of the crystal with respect to the direction of polarization is critical. Furthermore, the absorbed radiation is re-emitted with a high quantum efficiency; this resonance fluorescence has to be reduced by keeping the size of the filter small. Crystalline aqueous colloids of polystyrene spheres have been found to Bragg diffract visible light when the lattice parameters are comparable in length to the wavelength of the light.1° By controlling the angle of incidence of the scattered radiation to the crystalline colloid, the laser wavelength has been made to Bragg diffract and thus be rejected while the rest of the scattered radiation passes through the filter to a single-stage spectrograph. Crystalline colloidal Bragg diffraction filters have been used to obtain Raman spectra of semiconductor materials" such as crystalline silicon and gallium arsenide and of poly(propylene).12 An extension of the crystalline colloidal filter is the holographic Bragg diffraction filter which is made by recording a hologram in a dichromate gelatin/polymer emulsion between transparent ~ 1 a t e s . lThis ~ process generates a set of interference fringes which are recorded in the emulsion as successive planes of high and low refractive index separated by a distance that is half the wavelength of the recording light. When used as a Bragg diffraction filter, these can be configured to form very efficient notch filters. The characteristics and construction of holographic filters have recently been discussed by Morris.14 Organic dyes with absorption bands in the UV have been used as Rayleigh line rejection filters in time-resolved resonance Raman ~pectrometry.'~An organic dye with an absorption band in the visible region has also been used as a Rayleigh line rejection filter.16 Chevron-type Raman notch filters are commonly used in FT-Raman spectrometry. Notch filters are made of two identical dielectric bandpass filters which are mounted parallel at such an angle to the incoming parallel beam that the light is repeatedly reflected between the filters." The pass-band characteristics are such that at each reflection there is optimum transmission of laser light and effective reflection of Raman radiation. The filters described above have in some cases proved useful in reducing specular and Rayleigh scattering. However they all possess some disadvantage. The Bragg diffraction filters only work in the visible region, and like notch filters, their alignment is critical because the angle a t which they are oriented controls the wavelength of rejected radiation. Also, the observation of Raman spectral features at wavelengths corresponding to small energy shifta from the laser line is often made difficult due to filter attenuation which is spectrally too broad. The development of an efficient optical fiter to reject only the spectral region occupied by the laser line would eliminate the need for a multistage monochromator in routine Raman instrumentation and would permit the use of a more compact and less costly spectrometer with substantially improved optical throughput. Furthermore, such an optical fiter would be of use in FT-Raman spectrometry as well, allowing in both cases for low-frequency Raman spectral features to be observed. The intent of this study was to develop an optical fiiter for narrow-band laser scatter based on the resonance absorption of an atomic vapor. A cw Ti:Sapphire or pulsed dye laser, tuned to the atomic absorption line of suitable metal vapor (Rb at 794.76 nm and Hg a t 253.65 nm) was used for Raman excitation. The scatter from the sample was passed through a cylindrical cell containing this vapor before being dispersed by a single-stage monochromator and subsequently detected
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Figure 1. Experimental configuration for Raman spectrometry with rubidium vapor cell and cw Tksapphire laser. For pulsed Raman spectrometry with the mercury vapor cell, a XeCi excimer laser pumped dye laser was used with PMT detection. by a diode array or photomultiplier detector.
EXPERIMENTAL SECTION The cw experimental arrangement for Raman spectrometry is shown schematically in Figure 1. All lines of an argon ion laser (Model 2040, Spectra Physics, Mountain View, CA) were used to pump a single-mode, tunable, continuous-wave Titaniumsapphire laser in the ring configuration (Schwartz Electro-Optics, Inc., Orlando, FL), the output of which was directed through a focusing lens into a standard 1-cm quartz cuvette which contained the Raman sample. Scatter was collected at 90° and collimated through an electrically heated tube furnace which contained the metal vapor cell. The scattered light was then focused on the entrance slit of a 0.5-m focal length spectrograph (Model 1602, Spex Industries, Edison, NJ) and detected with an optical multichannel analyzer (Model TN-6500, Tracor Northern, Middleton, WI). The solid angle of the collected Raman radiation was matched to the acceptance of the spectrometer (fl6.9). The Tksapphire laser was tuned to the 794.76-nm rubidium line using the internal birefringent filter and etalon. The wavelength stability of the laser was sufficient to allow several hours of experimental work without adjustment. In this configuration, the laser spectral bandwidth was of the order of 0.01 pm with an output power of 300 mW. For the experimental measurements of laser absorption in the metal vapor cell as a function of cell temperature, the Raman sample cuvette was replaced with a small mirror at 4 5 O in order to direct the beam directly through the furnace. The commercially available metal vapor cell (Opthos,Rockville, MD) consisted of a Pyrex cylinder 7.6 cm long and 2.5 cm in diameter with Pyrex windows. An excess of rubidium metal was distilled into the evacuated cell such that sufficient metal would be present to give saturated metal vapor at all temperatures. Nitrogen gas at 500 Torr of pressure at room temperature was used as a fill gas in order to quench any resonance fluorescence from the metal. The cell was heated in a tube furnace (Model 55035, Lindberg, Watertown, WI) whose temperature could be controlled to within f l OC. The pulsed experimental setup was of the same general configuration. The laser in this case was a xenon chloride excimer laser (LPX-llOi, Lambda Physik, Gottingen, FRG) which was used to pump a dye laser (Model EPD-330, Lumonics, Inc., Ontario, Canada) which was frequency doubled to reach 253.65 nm. The spectral bandwidth of the doubled dye laser was about 5 pm, and the pulse energy at the sample cuvette was typically about 100 CJ. All optics, including the mercury vapor cell, were of fused silica. The mercury vapor filter was a laboratory-constructedcell of the same dimensions as the rubidium cell and also contained approximately 500 Torr of nitrogen. In the pulsed experiment, the scattered radiation was collected with a monochromator of 0.35-m focal length (ModelEU-700, Heath Corp.,Benton Harbor, MI) and was detected using a photomultiplier (R955, Hamamatsu Corp., Middlesex, NJ). The PMT photocurrent was terminated
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
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Flgure 2. Raman spectrum of carbon tetrachloride wRh cw laser excitation at 794.76 nm (a) without a Rb fitter and (b) with a Rb fitter. Spectrometer bandwidth was 20 cm-'.
into 1000 Q,passed through a 10-MHz bandwidth preamplifier (Model 4163,Evans Associates, Berkeley, CA), and monitored with a boxcar averager (SR 250, Stanford Research Systems,Palo Alto,CA) which WEE triggered by a photodiode which monitored the output of the excimer laser.
RESULTS AND DISCUSSION Variation of Absorbance with Temperature. Both Rb and Hg have sufficient vapor pressure at reasonable temperatures to form optically dense atomic populations. From the partial pressures18of metal vapors (Rb or Hg) in the cell at various temperatures, and thus at various number densities, the absorbances were calculated and found to agree with the measured values to within a factor of 2. For the purposes of the calculations, both lasers were assumed to be theoretical line sources. The absorption profiles of Rb and Hg in these cells have FWHM of 20 and 6 pm, respectively. These arise from the blending of the well-known Doppler and collisionally broadened hyperfine components. Such calculations required the absorption oscillator ~ t r e n g t h s 'and ~ the effective line widthsmas well as the ground-state number density. The lack of more precise agreement between calculated and measured absorbances is attributable to uncertainties in the absorption line profiles, especially with respect to the hyperfine structure and, in the case of Hg, to the pseudocontinuum nature of the laser spectral profile (5 pm). Nevertheless, it is evident that these filters at temperatures above 373 K provide extremely high absorbances (>8) for these two laser sources. Raman Spectra with Rb Metal Vapor Cell. The cw Raman spectrum of CCll with excitation at 794.76 nm with no metal vapor cell is shown in Figure 2a. Figure 2b shows a second spectra taken with the Rb cell in place at 280 OC. The stray light resulting from the Rayleigh and specular scatter is intense and broad, eliminating Raman shifts below about 70 cm-'. Raman peaks at 218 and 314 cm-l are clearly present in both Figures 2a and 2b. The degradation in signal
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Raman spectrum of d i d potassium nitrate with pulsed laser excitation at 253.65 nm (a) without a Hg filter and (b) with a Hg filter. Figure 4.
and signal-to-noise when the Rb cell is in place is a result of discoloration of the metal vapor cell windows with use at
Anal. Chem. 1992, 6 4 , 967-972
temperatures above 200 "C. The cause of the discoloration is not known but resulted in the need to heat the vapor cell to successively higher temperatures each time it was used in order to totally absorb the laser scatter. Raman Spectra with the Hg Vapor Cell. The pulsed Raman spectra of ethanol with excitation at 253.65 nm is shown in Figures 3a (no Hg cell) and 3b (Hg cell, 250 "C, in place). Just as in the case of the Rb filter, the laser scatter was completely eliminated by absorption in the metal vapor. However, in this case, the resolution of the monochromator system (1-nm spectral bandpass, about 150 cm-') was insufficient to resolve the two closely spaced peaks at 1100 cm-'. In Figurea 4a and 4b are shown the Raman spectra of a solid crystal of potassium nitrate with and without the use of the Hg vapor filter. In Figure 4a the laser scatter is so dominant that the Raman peaks at 1055 and 1350 cm-' are obscured. In Figure 4b, these two peaks are clearly evident after attenuation of stray light by the Hg filter. The small peak at 3600 cm-' in Figure 4a is an artifact and does not appear in Figure 4b.
CONCLUSIONS The Rb and Hg metal vapor filters were shown to be effective in eliminating laser scatter Raman spectra. It is clear that further work must be done to study the stability of these filters with time at elevated temperatures and to examine their practical use with more optimized Raman spectrometers. When used in conjunction with a single mode diode laser, locked, for example, onto the cesium transition at 852.10 nm, and a compact, high-efficiency spectrometer with diode array or CCD detection, such filters may make possible more sensitive and certainly more compact Raman instrumentation.
967
ACKNOWLEDGMENT This research was supported by a grant from the National Institutes of Health: NIH-5-RO1-GM 38434-04. REFERENCES Pelletier, M. J. Appl. Spectrosc. 1090, 4 4 , 1699. Woodward, L. A. I n Raman Spectroscopy: TheoryandFiactke;Vol. 2, H. A. Szymanski, ed.; Plenum Press: New York, 1970; Vol. 2. Long, D. A. Raman Spectroscopy; McGraw-Hill, Inc.: New York 1977. Devlln, G. E.; Davis, J. L.; Chase, L.; Geschwind, S. Appl. Phys. Len. 1971, 19, 138. Schoen, P. E.; Jackson, D. A. J. Phys. E. 1972, 5 . 519. Wail, K. F.; Chang, R. K. Opt. Len. 1986, 11, 493. Grossman, J. J. Fiiter Spectrograph. U.S. Patent No. 3,865,490, 1975. Huang, Y.; Yu, P. Y. Rev. Scl. Instrum. 1988, 59, 190. Gowers, C.; Hirsch, K.; Nleison, P.; Salzman, H. Appl. Opt. 1988, 27, 3625. Carison, R. J.; Asher, S. A. Appl. Spectrosc. 1984. 38, 297. Asher, S. A.; Flaugh, P. L.; Washinger, G. Spectroscopy 1986, 1 , 26. Flaugh, P. L.; O'Donneli, S. E.; Asher, S. A. Appl. Spectrosc. 1984, 38, 847. Carrabba, M. M.; Spencer, K. M.;Rich, C.; Rauh, D. Appl. Spectrosc. 1990, 4 4 , 1558. Yang, 8.; Morris, M. D.; Owen, H. Appl. Spectrosc. 1991, 45, 1533. Chou, P. T.; Studer, S. L.; Martlnez, M. L. Appl. Spectrosc. 1991, 45, 513. Hamaguchi, H.; Kamogawa, K. Appl. Spectrosc. 1988, 40, 564. Puppels. G. J.; Huizinga, A.; Krabbe, H. W.; de Boer, H. A.; Gijsbers, G.; de Mul, F. F. M. Rev. Scl. Instrum. 1990, 67, 3709. Smithells, C. J. Metals Reference Book, Vol. I I ; Butterworths: London, 1962; p 655. Radzig, A. A.; Smirnov, B. M. Reference Data on Atoms, Molecules and Ions; Springer-Verlag: Berlin, 1985. Alkemade, C. Th. J.; Hollander, Tj.; Sneliman, W.; Zeegers, P. J. Th. Metal Vapours in Flames; Pergamon Press: Oxford, 1982.
RECEIVED for review November 4,1991. Accepted January 27, 1992.
Capillary Array Electrophoresis Using Laser-Excited Confocal Fluorescence Detection Xiaohua C. Huang, Mark A. Quesada, and Richard A. Mathies* Department of Chemistry, University of California, Berkeley, California 94720 INTRODUCTION Capillary electrophoresis (CE) has found widespread application in analytical and biomedical research, and the scope and sophistication of CE is still rapidly advancing.'+ Gel-filed capillaries have been employed for the rapid separation and analysis of synthetic polynucleotides,6 DNA sequencing fragments,"-" and DNA restriction frag~nents.'~J~ Open-tube capillary electrophoresis has attained subattomole detection levels in amino acid separation^'^ and proven its utility for the separation of proteins, viruses, and bacteria.15 Separation of the optical isomers of dansyl amino acids has also been successfully demonstrated.16 Micellar electrokinetic capillary chromatography, isoelectric focusing, and on-column derivatization can all be performed on CE columns, demonstrating the utility of capillary electrophoresis as an analytical and micropreparative t001.~9~ The advantages of CE arise intrinsically from the use of a small inside diameter (20-200 pm) capillary. High electric fields can be applied along small diameter fused-silica capillaries without a significant increase in temperature. Since the electrophoretic velocity of the charged species is proportional to the applied field, CE can achieve rapid, highresolution separation. The reduced Joule heating in CE is a result of the low current passing through the capillary, the *To whom correspondence a n d r e p r i n t requests should be addressed.
large surface-to-volumeratio of the capillary channel, the use of thin capillary walls (50-150 Fm), and the high thermal conductivity of the wall material.' Although CE provides rapid analysis, thus far the total throughput is not high because only one capillary can be analyzed a t a time. Developing a method to increase the throughput of CE is a challenging and important task. One possible approach is to employ higher electric fields which would provide faster separations. Higher electric fields, however, often introduce overheating of the columns and column failure. Another way to increase the throughput is to run a large number of capillary separations in parallel. This approach uses an array of capillaries and is therefore called capillary array electrophoresis (CAE). CAE is potentially advantageous because the individual capillaries can be independently manipulated at the inlet, thereby facilitating rapid, parallel loading of multiple samples. In our approach, the capillaries are combined into a ribbon at the outlet for ease of parallel, on-column detection. In this way, a 2 order of magnitude increase in CE throughput should be achieved because hundreds of capillaries can be easily bundled for detection. An important problem confronting capillary array electrophoresis is detection. Since small amounts of sample are injected in a capillary, a high-sensitivity detection system is indispensable. Laser-excited fluorescence has proven to be a sensitive detection method in capillary electrophoresis and
0003-2700/92/0364-0967$03.00/00 1992 American Chemical Society
