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#Anal.Chem. 1986, 58, 1116-1119
Sedimentation Classification of Silver Colloids for Surface-Enhanced Raman Scattering Rong-Sheng Sheng,' Ling Zhu? a n d Michael D. Morris* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109
Colloidal silver sols for surface-enhanced Raman scattering are prepared by citrate or borohydride reduction of silver ions. The sols are fractionated by sedimentation. The fractions, each with a narrower sire distribution than the starting materlai, are classified by the intensity of the surface-enhanced Raman spectrum produced with a standard solution of crystal violet. The highest enhancement fractions are shown to provide crystal violet detection limits of 3 X lo-'' M. The separated fractions are stable for several weeks.
Surface-enhanced Raman scattering (SERS) has emerged as a potentially useful analytical tool (1-3). If silver is used as the enhancing metal, enhancement factors are typically 103-106. If a system is examined under electronic resonant conditions, the surface-enhancement and resonant enhancement are roughly multiplicative (4, 5 ) . In this case, SERS signals become quite large and detection limits can be in the range 10-9-10-12M (4,5). In the past, much SERS research has been devoted to understanding the phenomenon itself, and only recently has there been intense interest in development of analytically useful systems. Analytically attractive forms of silver include colloidal silver (5-7), silver island films (8), and silver films deposited on quartz or Teflon particles (3, 9, 10). In each case, the metal can be prepared independently of the sample. The sample can be mixed with a suspension of colloid or coated particles, or spotted on a plate containing silver island films (3, 7). Colloidal silver has been the most widely employed form of silver, possibly because its preparation requires no specialized apparatus. SERS signal intensities depend quite strongly on the local properties of the enhancing metal surface (11). For SERS use, colloidal silver should consist of aggregated particles, of average size 25-500 nm (3, 6-9). Several procedures for preparing silver colloids have been reported (12-14). These employ borohydride or citrate reduction of silver ion, often followed by heating or addition of a coagulant. However, it has been difficult to obtain reproducible enhancements or to prepare colloids that are stable for long periods of time using these methods. These problems impede interlaboratory comparison of SERS data and hinder widespread acceptance of SERS as a technique for trace analysis. In this communication we describe a practical method for preparing colloidal silver, which is stable for a t least three weeks and which yields intense and reproducible SERS spectra. Our technique is based on conventional chemical reduction, followed by sedimentation and classification of the most useful portions of a given batch. EXPERIMENTAL SECTION General Procedure. All chemicalswere reagent grade, where available, or highest commercially available purity. Type I reagent Permanent address: Center of Analysis and Measurement, Wuhan University, Wuhan, Hubei, China. Center of Analysis and Measurement,Wuhan University, Wuhan, Hubei, China.
grade water was used to prepare all solutions. Laboratory glassware was cleaned prior to use by sonication in aqueous detergent solution and repeated rinsing. Capillaries and microscope slides were cleaned by heating in a gas burner flame for approximately 1-2 min. Colloid Preparation. Colloidal silver sols were prepared by reaction of silver nitrate with sodium borohydride or sodium citrate (14). For borohydride reduction, 100 mL of 2.5 x IOm3 M AgNO, was added dropwise, with vigorous stirring, over a period of 20 min M NaBH, in an ice bath. The mixture to 300 mL of 2.0 X was then boiled for approximately 1 h to decompose excess borohydride and adjusted to 500 mL with distilled water. The = 384 nm. resulting colloids are brownish yellow, A, For citrate reduction, 500 mL of 1.0 X M AgNO, was M Na3C6H507 brought to boiling. To this, 10 mL of 3.4 X was added dropwise. The mixture was kept at boiling for about 1h. The final volume was adjusted to 500 mL with distilled water. = 406 nm. The resulting colloids are greenish yellow, A,, The colloids were transferred to graduated cylinders, covered, and allowed to fractionate by sedimentation over a 10-dayperiod. Successive aliquots of 25 mL were carefully drawn off from the cylinders. Crystal violet (Aldrich Chemical Co.) was dissolved in distilled M. Stock solutions water to prepare stock solutions of 2 X were diluted with distilled water, as required. Sample Preparation. Crystal violet solutions, 2 x to 2X M, were mixed with equal volumes of colloidal silver. Sampleswere drawn into capillaries, which were then flame sealed. To a clean microscope slide, 5 pL of silver colloid suspension was added with a micropipet. The suspensionwas dried by gentle heating. A 2.5-pL portion of crystal violet solution was dropped onto the colloid film and dried again by gentle heating, to produce a film whose diameter was about 0.5 cm. SERS Measurements. Spectra were obtained with a conventional Raman spectrometer, consisting of a Spex Instruments 1401 double monochromator and cooled RCA C31034A photomultiplier. Photon counting was used for data acquisition. Conventional90° illumination/examination geometry was used. Argon ion (Coherent, Innova 90) and helium-neon lasers (Spectra-Physics 125) radiation were used as excitation sources. Illumination intensities were about 10 mW at the sample. Data were taken at 100 cm-'/min scan rates, with 5-10 cm-' resolution and 1-s photon counter integration time. Scanning Electron Microscopy. Scanning electron micrographs were obtained with a Hitachi 650 instrument at 600X magnification. RESULTS AND DISCUSSION Crystal violet was chosen as a test system for this colloid preparation procedure, because its SERS has been previously characterized (15, 16). Figure 1 shows crystal violet spectra taken with 488-nm excitation with and without silver colloid. In the absence of silver colloids, only the strongest Raman bands are observable, even at low signal/noise ratio from solid crystal violet or from 2x M solutions at the excitation frequencies used here. Thus, all of the Raman signals observed are surface-enhanced signals. By comparison of the spontaneous Raman spectra a t millmolar concentrations to the surface-enhanced spectra a t micromolar concentrations, we estimate the signals for adsorbed crystal violet to be about lo6 more intense than the
0 1966 American Chemical Society 0003-2700/66/035S-1116$01.50/0
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
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signals for the bulk molecule. Figure 2 shows the absorption spectra for crystal violet, for the silver sol prepared by citrate reduction, and for the colloid/crystal violet mixture. The sol and crystal violet contributions to the mixture are apparent in the spectrum of the mixture. Figure 3 shows SERS for 1 X lo-’ M crystal violet as a function of exciting wavelength over the range 473-633 nm. All spectra were taken with approximately 20 mW power at the laser head, to give 10 mW at the sample. The excitation dependence of the 1175-cm-l line is plotted along with the absorption spectra, as Figure 2D. Data are not corrected for absorption of laser light or scattered light. On the basis of these data, 488 nm was chosen as the most practical excitation wavelength for further experiments.
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Flgure 3. Excitation wavelength dependence of SER spectra of CV: concentration, 1 X lo-’ M; laser power, 15 mW at sample; (A) 473 nm, (B) 476.5 nm, (C) 488 nm, (D) 496.5 nm, (E) 514.5 nm, (F) 633
nm. Excitation at 473 nm gives d signal that is approximately 25% higher. However, the 488-nm line is available from all argon ion lasers, even the smallest air-cooled models, and ancillary optics, such as spike and notch filters, are more widely available for 488 nm than for 473 nm. There are several intense bands of similar intensity in the crystal violet spectrum. At 488 nm the band at 1175 cm-l is the strongest. It is also well-resolved from other bands in the spectrum. Accordingly, we have chosen to use it for defining concentration and laser intensity dependences. We note that the excitation profiles of the various bands are not uniform. In particular, at 633 nm, the 210-cm-’ band is the most intense in the spectrum. That band was used by previous workers (15,16)who measured SERS spectra mostly at 633 nm. We find that the 1175-cm-l band intensity is also less dependent on solution conditions than is the lower frequency band, making it a more dependable indicator of concentration. Use of the higher frequency band is convenient in any case, since 210 cm-l lies on the edge of the Rayleigh scattering signal. From Figure 2D, it is clear that the maxima in the excitation profile are determined in large measure by the silver colloid. However, some contribution from electronic resonance is probable, since the data are mostly taken on the high energy side of the 545-nm band of crystal violet. A t 633 nm, quite close to the origin of the crystal violet electronic transition, the SERS intensity is small, demonstrating again that proximity to a plasmon resonance is necessary for best enhancement. It proved difficult to obtain stable SERS signals for crystal violet using the colloid preparation procedure described in ref 14, particularly if crystal violet concentration was below lo-’ M. Usually, the SERS signal was strongest about 2 days after the sample was prepared, and, if the capillary was stored vertically, the signal was strongest at the low end. This behavior is consistent with the observation that some coagulation is necessary for strong SERS (6). Addition of fluoride, chloride, bromide, iodide, or sulfate also caused the SERS signal to increase, as noted by others (4).Again, anions are functioning as coagulants.
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986 30
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Figure 4. Relative SER scattering intenslty (CV, 488 nm) for silver sol fractions as a function of sedimentation distance: A, sol prepared by cRrate reduction technique; 6,sol prepared by borohydride reduction technique.
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Figure 6. Concentration dependence of SER scattering intenslty for CV, 488 nm excitation: A, 1 X lo-' M; B, 1 X M; C, 1 X M; D, 1 X lo-'' M.
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Figure 5. Scanning electron micrographs of colloidal silver fractions: A, fraction 12; 6,fraction 7; C, fraction 6; D, fraction 3; E, fraction 1.
Sedimentation provides a simple and inexpensive method for obtaining coagulated and homogeneous colloids, suitable
for surface-enhanced Raman scattering. Figure 4 shows the crystal violet 1175-cm-l signal obtained from the sedimented fractions of silver colloids prepared by borohydride and citrate reduction and allowed to settle for approximately 15 days. The data are normalized to the response of the most active fraction. Scanning electron micrographs of colloids prepared by the citrate procedure are shown as Figure 5. From the figure, it is clear that particle sizes increase by coagulation and that the sedimentation procedure effective sorts the coagulated and uncoagulated silver. The top fraction, number 12, shows little coagulation. The highest enhancement fraction, number 7, is extensively coagulated but contains no bulk crystalloids. The fraction with the second highest enhancement, number
6, contains a few crystalloids in addition to aggregates. The heaviest fraction, number 1, is predominantly crystalline silver and shows low enhancement. Enhancement trends in the colloid fractions made by the two procedures are similar. In each case the lightest and smallest particles, occupying the upper 15% of the volume, show weak or no enhancement. The enhancement then increases rapidly with particle size (fraction), reaching a sharply defined maximum about halfway down the sedimentation flask. As the particle size increases, the enhancement decreases. The falloff is rapid for colloids prepared by the borohydride procedure. For the colloids prepared by the citrate procedure the decrease is less precipitous, and there is a broad region where the enhancement is more than 50% of the peak value. These data confirm the electron microscopy result that the citrate reduction procedure produces colloids with a fairly uniform particle size distribution, while the colloid produced by borohydride reduction is less homogeneous. Similar behavior occurs with gold colloids (17). Since the absolute enhancement obtained with citrate colloids is approximately 3 times higher than with borohydride colloids, this particle size distribution is not only more uniform but also closer to the optimum for excitation with 488-nm light. The highest and second highest enhancement fractions of the citrate and borohydride colloids were chosen to test the linearity and detection limits of SERS obtained with colloids prepared by this procedure. Figure 6 shows crystal violet spectra obtained with the highest enhancement citrate colloid at concemrations between lo-' and 10-lo M, Working curves obtained for the citrate colloids were linear (relative standard deviation 5-7% for the best fraction, 12% for the next best) over the range 1 X to 1 X M, and reached a maximum around 3 X M, reflecting saturation of the available interaction sites (15). Above this value, a slight decline was observed, as absorption of laser light became significant. With colloids prepared by the borohydride procedure, working curves were linear (relative standard deviation 12-18%) from about 1 X M to 1 X lo4 M, with saturation occurring around 2 X lo4 M. With both types of colloid, crystal violet concentrations about 1 X M caused further
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Anal. Chem. 1986. 58. 1119-1123
coagulation of the colloids, leading to a decreased signal and, ultimately, to formation of visible particles. For the highest enhancement silver colloid a detection limit ( S I N = 2) of 3 X lo-" M is obtained. A similar series of measurements was made for citrate colloid deposited on glass microscope slides. In this case detection limits are approximately 1pg of crystal violet, and working curves are linear (relative standard deviation 2%) to at least 1 ng.
CONCLUSIONS The citrate reduction of silver ion is superior to the borohydride method for preparation of silver colloids for surface-enhanced Raman spectroscopy. The fraction of the preparation that is coagulated to the required extent is greater, and the final colloid shows a more uniform particle size distribution. Sedimentation provides a simple and effective method for selecting the fraction of a colloid batch that is useful for SERS. Sedimentation appears to be about as effective as ultracentrifugation for isolation of the active fraction of the colloid. The colloids prepared by this technique can be used for measurement of samples in solutions or for samples spotted onto a glass slide. The latter procedure may prove appropriate for samples that are dissolved in or extracted into water-immiscible solvents. The detection limits obtained in this study, and similar results obtained for rhodamine 6G (6),place SERS among the most sensitive available analytical techniques for molecules in solution, not far behind laser-excited fluorescence. The 3-10 cm-' bandwidths of Raman lines and the qualitative information inherent in a Raman spectrum suggest that SERS may play an increasing role in trace analysis.
Registry No. AgN03, 7761-88-8; NaBH,, 16940-66-2;Na3CSH507,68-04-2; Ag, 7440-22-4;crystal violet, 548-62-9. LITERATURE CITED (1) Gantner, E.; Steinert, D.; Reinhardt, J. Anal. Chem. 1985, 5 7 , 1658-1662. Jennings, C.; Aroca, R.; Hor, A,-M.; Loutfy, R. 0.Anal. Chem. 1984, 56. 2033-2035 -___ Vo-Dinh, T.; Hiromoto, M. Y. K.; Begun, G. M.; Moody, R. L. Anal. Chem. 1984, 5 6 , 1667-1770. Siiman, 0.;Lepp, A.; Kerker, M. J . fhys. Chem. 1983, 8 7 ,
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(5) Hildebrandt, P.; Stockburger, M. J . fhys. Chem. 1984, 8 8 , 5935-5944. (6) Creighton, J. A. In "Surface Enhanced Raman Scatterlng"; Chang, R. K., Furtak, T. E., Eds.; Plenum Press: New York, 1982; pp 315-337. (7) Kerker, M.; Wang, D.-S.; Chew, H.; Siiman, 0.; Bumm, L. A. In "Surface Enhanced Raman Scattering"; Chang, R. K., Furtak, T. E., Eds.; Plenum Press: New York, 1982; pp 109-128. (8) Ritchie, G.; Chen, C. Y. In "Surface Enhanced Raman Scattering"; Chang, R. K., Furtak, T. E., Eds.; Plenum Press: New York, 1982; pp 361-379. (9) Goudennet, J. P.; Begun, G. M.; Arakawa, E. T. Chem. fhys. Lett. 1982, 92, 197-201. (10) Meier, M.; Wokaun, A.; Vo-Dinh, T. J . Chem. Phys. 1985, 89, 1843-1846. (11) Birke, R. L.; Lombardi, J. R. I n "Advances In Laser Spectroscopy"; Garetz, B. A., Lombardi, J. R., Eds.; Heyden and Son: London, 1982; VOI. 1, pp 143-152. (12) Creighton, J. A.; Blatchford, C. G.; Aibrecht, M. G. J . Chem. Soc., Faraday Trans. 2 1979, 75, 790-798. (13) Fabrikanos, A.; Athanassiou, S.; Lieser, K. H. 2. Naturforsch., B : Anorg. Chem., Org. Chem., Biochem., Biophys., Biol. 1963, lBB, 612-617. (14) Lee, P. C.; Meisel, D. J. J . fhys. Chem. 1982, 86, 3391-3395. (15) Tran, C. D. Anal. Chem. 1984, 5 6 , 824-026. (16) Tran, C. D. J . Chromafogr. 1984, 292, 432-438. (17) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss. Faraday Soc. 1951, 1 1 , 55-75.
RECEIVED for review July 19, 1985. Accepted December 26, 1985. Financial support was provided by the National Science Foundation through Grant CHE-8317861.
Detection of Nitro Polynuclear Aromatic Compounds by Surface-Enhanced Raman Spectrometry Paul D. Enlow, Milan Buncick, Robert J. Warmack, and Tuan Vo-Dinh*
Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
The various nitro polynuclear aromatic compounds, including 1-nitropyrene, 9-nitroanthracene, 2-nitronaphthalene, and 2nitrofiuorene have been investigated by use of the surfaceenhanced Raman scattering (SERS) spectrometry technlque. Silver-coated substrates consistlng of latex spheres on glass and filter paper and prolate SIO, posts on quartz were used. The llmit of detectlon of 1-nitropyrene was found to be 0.3 ng. The SERS signals were enhanced over conventional Raman signals by a factor of 5 X 10' to 9 X 10' for 1-nitropyrene adsorbed on various SERS-active substrates. The production and practlcality of SERS-active substrates with glass, paper, and quartz supports are discussed.
Nitro polynuclear aromatic (nitro-PNA) compounds have recently received intensive interest. These compounds are often produced in atmospheric reactions in PNAs with NO, or during incomplete combustion in automotive engines. 0003-2700/86/0358-1119$01.50/0
Nitro-PNA species have been detected in a variety of products including ambient particulates (1-3), diesel exhaust particulate ( 4 , 5 ) ,and carbon black and xerographic toners (6, 7). Correlations between the presence of nitro-PNAs in environmental extracts and mutagenic activity have been reported in a number of studies (8,9). A recent study has also reported the carcinogenicity of some of the nitro-PNAsin rats (10). Due to the biological and environmental importance of these nitro-PNA compounds, a variety of analytical techniques have been developed for the identification and quantification of these species. Most of these techniques involve chromatographic methods such as gas chromatography-mass spectrometry (4), glass capillary gas chromatography (11),highperformance liquid chromatography (12), and thin-layer chromatography (13). In this paper we evaluate the detection technique based on the surface-enhanced Raman scattering (SERS) technique using silver-coated solid substrates. A number of observationshave recently indicated enhancement in the Raman scattering efficiency by factors of 103-106where 0 1986 American Chemical Society
