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Anal. Chem. 1990, 62, 678-680
Surface-Enhanced Raman Spectroscopy at a Silver Electrode as a Detection System in Flowing Streams Neil J. Pothier and R. Ken For&* Department of Chemistry, University of Rhode Island, Kingston, Rhode Island 02881
An analytical appkatlon for surface-enhanced Raman spectroscopy at a silver electrode is described. Raman spectra of several DNA bases have been recordsd In a 30-pL flow cell under flowing conditions. An optical multichannel analyzer allowed high-quality spectra to be recorded with Integration times of less than 5 8. The Raman Intensity vs concentration yields a monotonic relationship over 4 orders of magnitude. Limits of detection for adenine, thymine, and cytosine were 175,233, and 211 pmd, respectively. The samples could be rapldly desorbed off the surface of the electrode in less than 10 s. The technique shows promise as a solute property detector for high-performanceUquM chromatography and fbw injection analysls.
INTRODUCTION Surface-enhanced Raman spectroscopy (SERS) has received considerable interest since first reported in 1974 by Fleishmann and co-workers (1). Several articles appearing in the literature are directed toward a better theoretical understanding associated with the giant enhancement in the Raman spectra of molecules resulting from interactions at or near a SERS active surface (2-4). SERS has been studied most extensively in silver sols and films and a t roughened silver and gold electrodes (5-10). Recent applications of SERS include Ag microspheres and Ag-coated filter paper (I1,12). Excellent reviews on the theory and application of SERS have recently appeared (13,14).However, only a few articles have appeared detailing SERS as a useful analytical technique in flowing streams ( I 5-1 8). Our research is directed toward the development of SERS as a detector for high-performance liquid chromatography (HPLC) and flow injection analysis (FIA). From previous work we have demonstrated the ability to collect SERS spectra of 0.05 M pyridine in aqueous media under dynamic conditions in a 200-pL Raman flow cell (17). In a three-electrode 30-pL flow cell under flowing conditions we have demonstrated the ability to rapidly adsorb and desorb selected DNA bases by controlling the applied potential a t a silver electrode. In addition we have successfully demonstrated with an optical multichannel analyzer (OMA) that high-quality spectra spanning a 800-cm-’ window can be collected in less than 5 s, yielding the vibrational molecular fingerprint of solutes eluting through the cell. EXPERIMENTAL SECTION Instrumentation. The SERS flow cell utilizes a three-electrode system consisting of a 4 mm 0.d. silver working electrode, a platinum foil auxiliary electrode, and a saturated calomel electrode (SCE) mounted in a Plexiglas cell constructed in our laboratories. See Figure 1. The silver working electrode consists of 4 mm diameter Ag foil which has been epoxy cemented into a 4 mm glass tube and soldered to a copper lead. The electrode is easily removed for polishing or alignment from ita threaded housing. Two standard HPLC fittings act as inlet and exit ports and a glass microscope slide provides a window for spectroscopic detection. The platinum (1 mil thick) foil auxiliary electrode
surrounds the working electrode to minimize variations in the potential field. A gasket provides a nominal cell volume of 30 ML. The cell WBS evaluated in two different spectrographic systems. System 1 utilized a Jarrell-Ash 3/4-m Czerny-Turner single monochromator with a 1180 lines/” grating blazed at 500 nm. Incident 514.5-nm radiation from a Spectra-Physics Model 2000 Art ion laser operated at 20 mW at the cell served as the source. A camera lens collected and focused the scattered light onto the entrance slit of the monochromator. A Shott OC 550 cutoff filter was used to attenuate the incident laser radiation and allowed for a window from 900 to 1700 cm-’ to appear across the focal plane of the polychromator. Because of the spectral limits of the cutoff filter, Raman bands below about 900 cm-’ could not be observed. Mounted at the exit focal plane is an OMA (Princeton Instruments Model IRY-700 Demo) regulated by a Model ST-120 controller. The OMA was operated at -20 OC. Data from the OMA was controlled and stored on a Dell Model 220 microcomputer using DMA software obtained from Princeton Instruments. A potentiostat (PARC Model 364) was used to maintain POtentiostatic control of the cell and to electrochemically roughen the silver electrode surface by oxidation-reduction cycles (ORC’s). For SERS to occur the surface of the electrode must be roughened from an ORC. The silver electrode is first polished with 6-, then 3-, and finally 1-pm diamond paste and rinsed with distilled water, methyl alcohol, and again with water and placed in the SERS cell. The ORC’s consist of stepping the voltage to +0.2 V vs SCE in order to pass between 100 and 150 mC/cm2 and then stepping the voltage to -0.6 V where the greatest enhancement for pyridine and other nitrogen-based compounds have been reported (18,19). This procedure yielded a very reproducible surface. The solutes are injected into the flowing solvent, and when the solutes entered the cell another 1-2 s ORC is performed. Preliminary results showed that the greatest enhancement was obtained when an ORC was performed while the solute was present in the cell. An Altex Model llOA HPLC pump was used to maintain a constant flow of solvent through the cell. The mobile phase consisted of 0.1 M KC1 in 0.01 M KH2P04buffer adjusted to pH 7.00 with KOH. Because the most intense Raman bands for the DNA bases examined in this study all lie below 900 cm-’, the single spectrograph with cutoff filter prevented their study on that system. Therefore, a second instrument configuration utilizing a Spex 1401 double monochromator with the same collection optics as described above was employed. The OMA was mounted at the exit slit where the conventional photomultiplier tube is housed. With this confiation, we had adequate stray light rejection. However, only a 50-70-cm-’ optical window could be viewed across the face of the OMA because the intermediate and exit slits of the Spex were not removed. Since the objective was to evaluate the time response under potentials leading to selective adsorption or desorption of the analyte to the Ag electrode, this was not a serious limitation. Limits of detection studies were evaluated with this arrangement. All chemicals used were ACS reagent grade and used without further purification. Adenine, cytosine, and thymine were obtained from Sigma Chemical Co. (St. Louis, MO). The mobile phase consisted of 0.1 M KCl and 0.01 M KH2P04(Fisher Scientific Co.) buffered at pH 7.00 and was used to dissolve the DNA bases. A solution of 0.05 M pyridine (Fisher) was also prepared in this mobile phase. Serial dilutions of adenine, thymine, and cytosine were prepared from 5.88, 7.77, and 7.02 mM stock solutions, respectively.
0003-2700/90/0362-0678$02.50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 7, APRIL 1, 1990
679
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Flgure 1. 30-pL SERS flow cell: (A) metal window frame; (B) rubber gasket; (C) 2 cm X 2 cm X 1 mm glass window; (D) platinum foil auxiliary electrode; (E) 30-pL gasket; (F)Plexiglas cell; (G) exit tubing; (H) calomel reference electrode; (I) inlet tubing; (J) silver working electrode; (K) rubber O-ring: (L) silver wire lead; (M) 4 m m glass tubing; (N) knurled nut; (0) copper lead.
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Flgure 3. 400-wL injections of 5.10 mM adenine (0)detected at 732 cm-', 9.83 mM cytosine (A)detected at 796 cm-', and 4.96 mM thymine (0) detected at 776 cm-'. The upward pointing arrows ( f )
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Flgure 2. 200-pL repeat injections of 0.05 M pyridine injected into phosphate buffered 0.1 M KCi flowing at 1.0 mL/min.
indicate the potential step to -0.6 V, the enhancing potential. The downward pointing arrows (1)indicate the potential step to -1.3 V, the desorbing potential. I
I
RESULTS AND DISCUSSION For SERS to be of practical use as a detector for highperformance liquid chromatography (HPLC) and flow injection analysis (FIA) it is necessary that the analyte be rapidly and selectively removable from the electrode surface. As can be seen in Figure 2 with 200 pL repeat injections of 0.05 M pyridine into the phosphate-buffered KC1 flowing at 1.0 mL/min, we can repetitively cycle and analyte on and off the electrode with very rapid time response. At the time of injection, the electrode was subjected to a rapid 1-s ORC; the potential was then manually adjusted to -0.6 V vs SCE and held there for 20-30 s and was then manually adjusted to -1.2 V to desorb the analyte. Raman spectra were obtained continuously during this time and either stored in memory or written to disk. As can be seen, the reproducibility from one injection to another is excellent. Also, it is clear that the analyte can be completely removed from the electrode in less than 10 s under flowing conditions. The signal was integrated on the face of the OMA for 5 s and every other frame was stored. To verify the efficiency of removal, on selected injections, after desorbing the analyte, the potential was manually adjusted back to -0.6 V after about 30 s and no evidence of any pyridine could be detected. Illustrated in Figure 3 are the time-dependent traces for 400-pL injections of adenine, cytosine, and thymine into the phosphate buffered 0.1 M KC1 flowing at 1.0 mL/min. A t the time of injection, the electrode was subjected to a 1-s ORC, the potential was manually adjusted to -0.6 V, two or three frames were integrated, and then the potential was manually
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Flgure 4. Duplicate 400-pL injections of 5.10 mM adenine injected into phosphate buffered 0.1 M KCI flowing at 1.0 mL/min, detected at 732 cm-'. The upward pointing arrows (t) indicate the potential step to -0.6 V, the enhancing potential. The downward pointing arrows (1) indicate the potential step to -1.3 V, the desorbing potential.
adjusted to -1.3 V to desorb the analyte. It proved necessary to step to a more cathodic potential to efficiently remove the DNA bases from the surface of the electrode. Adenine was found to give somewhat higher enhancement than either cytosine or thymine. If one closely examines Figure 3, it can be seen that the majority of the material can be eluted from the electrode in about 10 s. The downward pointing arrows indicate the point where the potential was stepped to -1.3 V, the desorbing potential. The delay in analyte removal is in part attributed to a large (400 pL) dead volume between injector and cell. With this taken into account, the majority of the analyte is removed from the electrode in a fraction of a second. For each run, the spectra were integrated for 5 s, and every other frame was stored in memory. As can be seen from Figure 4, repeat injections of adenine showed a reproducibility of 5%.
680
Anal. Chem. 1990, 62,680-683
To determine the sensitivity of SERS at a Ag electrode for selected DNA bases, standard solutions of adenine were injected under stopped flow conditions and the intensity of the 732-cm-' band was integrated for 5 s. Stopped-flow conditions were utilized because of the difficulty in coordinating manual adjustment of the potentiostat and the applied potential as the analyte entered the cell. The intensity showed a monotonic relationship with concentration and a detection limit of 510 pmol. In addition, under stopped-flow conditions, adenine, thymine, and cytosine limits of detection were evaluated with a photomultiplier tube (RCA C31034-RF) and yielded 175, 233, and 211 pmol, respectively. Even though the cell volume reported here is at least a factor of 5-10 smaller than any other design reported in the literature, it is not optimum, and only a very small fraction of the total cell volume is being interrogated by the laser beam. Also, since SERS is a surface phenomenon, a very small fraction of the total analyte in the cell is being probed. With further improvements in cell volume and the ratio of cell volume to electrode area, it should be possible to considerably reduce the limits of detection below those reported here. This research has clearly proven the rapid response to adsorption and desorption controlled by potential modulation at the silver electrode. The technique yields good reproducibility. The concentration and volume ranges are rapidly approaching those necessary for modern liquid chromatographic systems. The need for a solute property detector yielding qualitative and quantitative information is of extreme importance in complex biochemical separations, and we feel SERS at a Ag electrode may prove beneficial as a detector for HPLC and FIA. Continuing research in this laboratory is directed toward the utilization of gold electrodes, which have proven a very stable SERS active substrate (20-22). In addition further
miniaturization of the cell and computer control of the potentiostat are presently under development.
ACKNOWLEDGMENT We thank Charles Nittrouer and Princeton Instruments for the use of the optical multichannel analyzer and associated data acquisition hardware and software. LITERATURE CITED Fleishmann, M.; Hendra, P. J.; McQuillan, A. J. Chem. Phys. Lett. 1974, 26, 163. Surface Enhanced Raman Spectroscopy; Chang, R. K., Furak, T. E.; Eds.; Plenum Press, New York, 1982. Fleishman, M.; Hill, 1. R. Compr. Treatise Electrochem. 1984. 8 , 373. Jeanmire, D. L.; Van Duyne, R. P. J. Electroanal. Chem. Interfacial Electrochem. 1977, 84, 1. Suh, J. S.; Moskovits, M. J. Am. Chem. SOC. 1988, 108, 4711. Moskovits, M.; Suh, J. S. J. Fhys. Chem. 1984, 88, 1293. Joo, T. H.; Kim, M. S. Chem. Fhys. Lett. 1984, 112, 65. Chou, Y. C.; Liang, N. T.; Tse, W. S. J. Raman Spectrosc. 1986, 17, 481. Gao. P.;Gosztola, D.; Leung. L.; Weaver, M. J. Elechoanal. Chem. Interfacial Electrochem. 1987, 233, 21 1. Weaver, M. J.; Hupp, J. T.; Barz, F.; Gordon, J. G., 11; Philpolt, M. R. J . Electroanal. Chem. Interfacial Electrochem. 1984, 160, 321. Vo-Dinh, T.; Uzeil, M.; Morrison, A. L. Appl. Spectrosc. 1987, 41. 4. Lasema, J. J.; Campiglia, A. D.; Winefordner, J. D.Anal. Chem . Acta 1988, 208, 21. Garrell, R. L. Anal. Chem. 1989, 61, 401A. Birke, R. L.; Lombardi, J. R. Spectrmlectrochemisby, Theory and Practice; Gale, R. J., Ed.; Plenum hess: New York, 1988. Ni, F.; Thomas, L.; Conon, T. M. Anal. Chem. 1989, 6 1 , 888. Berthod, A.; Laserna, J. J.; Winefordner, J. D. Appl. Spectrosc. 1987, 4 1 , 1137. ForcB, R. K. Anal. Chem. 1989, 60, 1987. Freeman, R. D.; Hammaker, R. M.; Meloan. C. E.; Fateley, W. G. Appl. Spectrosc. 1988, 42, 456. Leung, L. W. H.; Weaver, M. J. J. Am. Chem. Soc.1987, 109, 5113. Ganell, R. L.; Beer, K. D. Spectroch/m. Acta 1988, 43, 617. Patterson, M. L.; Weaver, M. J. J. Fhp. Chem. 1985, 89, 1331.
RECEIVED for review August 11, 1989. Revised manuscript received December 26, 1989. Accepted January 2, 1990.
Laser-Induced-Fluorescence Detection of Sodium Atomized by a Microwave-Induced Plasma with Tungsten Filament Vaporization Yuji Oki,* Hisanori Uda, Chikahisa Honda, Mitsuo Maeda, Jun Izumi,' Takashi Morimoto,' and Masazumi Tanoura'
Department of Electrical Engineering, Kyushu University, Hakozaki, Fukuoka 812, Japan
The laser-lnduced-fluorescence(LIF) technique is applied to the detection of Na atoms in pure water for a concentration range down to pg/cms. Na compounds are dissociated by a mlcrowave-krduced plasma of He wlth a tungsten filament vaporization system. CaUbrating the absolute denslty at the observing region, the efficiency of thk atomizer Is estimated. I t Is shown that the atomizer can generate hlgh atomic denslles by fHament heating. For example, the number denslty reaches lo7 atoms/cms for a lO-wL sample of 1 pg/cm3.
The sensitivity of laser-induced-fluorescence (LIF) spectroscopy with a tunable dye laser is excellent especially for *Nagasaki R & D Center, M i t s u b i s h i H e a v y Industries, Akunoura-machi, Nagasaki 850-91, Japan
Ltd.,1-1
the detection of atomic species. In an extreme case, the detection of individual atoms is possible when using a vapor cell or an atomic beam (1-4). Applications of these techniques to the atomic analysis are expected to provide sensitivity much higher than traditional methods. The first application of LIF to the frame atomic fluorescence spectroscopy was reported Fraser and Winefordner in 1971 (5, 6). Detection limits of metals (Ca, Na, Sr, Mg, etc.) lower than 1 ng/cm3 were reported by Weeks et al. (7);furthermore, lower detection limits were obtained with a graphite furnace. For example, Hohimer and Hargis obtained 20 pg/cm3 in Cs (8) and 0.5 pg/cm3 in Ti (9). In LIF, it is not difficult to detect atoms with concentrations as low as 105-106 atoms/cm3 in an atomic vapor cell. The potentiality of the LIF method in the detection sensitivity is extremely large. However, the atomizer usually limits it.
0003-2700/90/0362-0680$02.50/00 1990 American Chemical Society
