3712 Langmuir 1995,11, 3712-3720

Characterization of the Surface of a Citrate-Reduced Colloid Optimized for Use as a Substrate for

Surface-Enhanced Resonance Raman Scattering

C. H. Munro, W. E. Smith," M. Garner, J. Clarkson, and P. C. White

Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 IXL, U.K.

Received February 28, 1995. In Final Form: June 26, 1995@

Citrate-reduced colloids can be used to give reproducible, sensitive, and selective analysis by surface- enhanced (resonance) Raman scattering (SE(R)RS). Control of the chemistry at the colloid surface is essential to realize the potential ofthis method. This study is aimed at understanding the surface chemistry in aqueous solutions, characterizing the nature of the final surface, and developing a robust method for SE(R)RS analysis at the surface. An optimized procedure for the reduction of silver nitrate with trisodium citrate is described. Visible absorption and photon correlation spectroscopies of colloid formation indicate that the initial reduction of Ag' to AgO occurs within 2 min of citrate addition, and the initial particles formed are large (60-80 nm) and polydisperse. Subsequent heating initially provides a less polydisperse mixture of 20-30 and 40-50 nm particles and finally an approximately monodisperse distribution of smaller particles (-27 nm). Solution NMR studies of the colloidal suspension indicate the presence of citrate and its decomposition products, acetoacetic acid and formate in solution throughout colloid formation. Raman scattering from aggregated aliquots of colloid indicates two forms of citrate depending on the stage of preparation, but neither acetoacetic acid nor formate is detected as being adsorbed at the silver surface. The final, approximately monodisperse particles are believed to be stabilized by a surface layer of silver citrate, with pendant negative groups. The colloids are stable for over 2 months. The SE(R)RS effect requires controlled aggregation of the colloid. The aggregation process is generally induced by the addition of acid or activating ions, for example, C1- or I-. Aggregation with acid (HN03) and with poly(L-lysine) and ascorbic acid are compared. The poly(L-lysine) method is more effective, enhancing the monodispersity of colloidal aggregates. The reproducibility of SERRS (relative standard deviation (RSD) < 5%) is acceptable for analytical purposes, whereas that from aggregation with acid (HN03) (RSD = 18.5%) is not. Futhennore, at low analyte concentrations, SE(R)RS from both the analyte and the citrate layer are observed on aggregation with nitric acid. However, SE(R)RS is only observed from the analyte on aggregation with poly(L-lysine) and ascorbic acid. The advantages for trace analysis of anionic, neutral, and cationic species of using reagents which alter surface charge and dielectric constant are illustrated.

Introduction The potential of surface-enhanced Raman scattering

(SERS) as an analytical tool is well es tabl i~hed. l -~~ The sensitivity resulting from an enhancement in scattering

* Author to whom correspondence should be sent. @ Abstract published in Advance A C S Abstracts, September 15,

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(7) Garrell, R. L.; Anal. Chem. 1989, 61, 401. (8) Gouveia, V. J. P.; Gutz, I. G.; Rubim, J. C. J . Electroanal. Chem.

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(10) Laserna, J. J. Anal. Chim. Acta 1993, 283, 607. (11) Montes, R.; Laserna, J . J. Analyst 1990, 115, 1601. (12) Montes, R.; Contreras, C.; Ruperez, A.; Laserna, J. J.Anal. Chem.

(13) Munro, C. H.; Smith, W. E.; Armstrong, D. R.; White, P. C. J .

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0743-74631951241 1-3712$09.00/0

by a factor of approximately 105-106, the detailed structural information, and the selectivity resulting from surface orientation enable the characterization of mol- ecules adsorbed at submonolayer coverage onto roughened metal surfaces. Furthermore, where the excitation wave- length is coincident with molecular resonance, an ad- ditional enhancement is obtained from the surface- enhanced resonance Raman scattering (SERRS). The selective enhancement associated with resonance Raman scattering is retained, giving good molecular specificity, free from interference from non-SERRS-active contami- nants. Fluorescence may be quenched and good signals recorded over a greater range and at much lower con- centrations. In addition, it is less sensitive to surface orientation and is more robust than SERS with good signals obtained from very small amounts of material.13 Thus, SERRS can be used for sensitive qualitative and semiquantitative analysis. However, before SERRS can fulfill its potential as an analytical method, a well- characterized SERS substrate and a reliable procedure are required which will yield reproducible results.

Surface-enhanced Raman scattering (SERS) was first observed from pyridine on an electrochemically roughened

(21) Taylor, G. T.; Sharma, S. K.; Mohanan, K.Appl. Spectrosc. 1990,

(22) Torres, E. L.; Winefordner, J. D. Anal . Chem. 1987, 59, 1626. (23) Tran, C. D. J . Chromatogr. 1984,292, 432. (24) Tran, C. D. Anal. Chem. 1984, 56, 824. (25) Vo-Dinh, T.; Alak, A.; Moody, R. L. Spectrochim. Acta 1988,

(26)Vo-Dinh, T.; Houck, K.; Stokes, D. L. Anal. Chem. 1994, 66,

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0 1995 American Chemical Society

Surface of a Citrate-Reduced Colloid

silver electrode.28 Although silver electrodes provide an effective SERS substrate with unique advantages for electrochemical studies, the surface roughness is difficult to control and to characterize, and the reproducibility of scattering enhancement is poor. Over the last 20 years, several substrates for SERS and SERRS have been examined for their analytical potential. These include a limited number of metals (Ag, Au, Cu, Al, In, Li, and Na) in various morphologies, e.g., colloidal suspensions, grat- ings, island films, and films deposited on paper substrates and quartz or Teflon particles or The most common substrates used are colloidal suspensions of silver. They are easily prepared, can be well characterized, and require minimum sample handling. The two colloids used most widely are prepared by the reduction of silver nitrate with borohydride and with itr rate.^^,^^

Many studies have already been carried out to char- acterize these ~ o l l o i d s . ~ ~ , ~ ~ , ~ ~ ~ ~ ~ Reduction with citrate produces a more uniform particle size distribution than that produced with borohydride. In addition, the stability with time and the magnitude of enhancement is greater for the citrate-reduced The stabilizing effect of protective surface layers on metal particles has been well d o c ~ m e n t e d . ~ ~ The stabilization of a Carey-Lea by a surface layer of citrate adsorbed by flocculation of the colloidal particles with trisodium citrate solution has been reported by Siiman et al.36 The stability of the citrate- reduced colloid may be attributed to the formation of such a layer during the reduction procedure.

The use of SE(R)RS in analytical procedures requires control ofproperties such as adhesion, surface orientation, and biocompatibility. For example, in recent studies of azo dyes in dilute solution, only a limited number of dyes were adsorbed satisfactorily a t the surface of the citrate- reduced colloidal particles.13J4 Neutral and cationic azo dyes were readily adsorbed onto the colloid surface, and strong SERRS resulted on aggregation. However, neither adsorption nor SERRS was observed for anionic azo dyes. With the knowledge that the colloid surface layer was negatively charged, it was shown that the addition of the cationic poly(amino acid) poly(L-lysine) to the colloidal suspension provides a more general method. It is pos- tulated that there is adherence of the protonated amino residues of poly(L-lysine) to the anionic species and to the negatively charged colloid surface layer. Good SERRS from 20 anionic azo dyes was obtained, and the molecular specific nature of the scattering enabled discrimination between the dye structures, including structural isomers, for the first time. In addition, linearity in a plot of con-

, Langmuir, Vol. 11, No. 10, 1995 3713

centration versus scattering iniensity was observed at low solution concentrations (<3 x M), supporting the application of SERRS from citrate-reduced colloids for qualitative and semiquantitative analysis of trace amounts (2300-500 pg) of acidic monoazo dyes. Fur- thermore, the effect of the different reduction procedures on the nature of the surface at which the analyte is adsorbed is evident from results of a study of the enzyme cytochrome P-450.44 On adsorption at the surface of a borohydride-reduced colloid, the enzyme changed spin state. However, the spin state is conserved if a citrate- reduced colloid is used and the appropriate conditions for the conservation ofthe enzyme in solution are maintained throughout. This observation was attributed to the presence of the protective surface layer formed on citrate reduction, which provides an organic surface for attach- ment to the protein by surface charges, preventing direct silver-protein interaction.

Thus, the advantages of the citrate-reduced colloid and the importance of an understanding of the nature of the surface are evident. However, the problem which is indicated by the literature is that the preparation of the colloid has proved difficult to reproduce in some labora- tories. The absorbance spectra ofthe colloids vary greatly. The maximum absorbance (Amm) ranges between 406 and 450 nm (generally -420 nm), and the full width at half- height (fwhh) ranges between 115 and 300 nm, indicating notable differences in particle size distribution. In ad- dition, different results are obtained in that not all laboratories can observe the signal attributed to the organic layer. The extension of SE(R)RS for routine analysis requires the optimization of the reduction procedure with respect to both SE(R)RS intensity and reproducibility, and the intention to modify the surface by addition of other ligands requires a detailed study of the colloid preparation and the chemistry of the surface formed.

The correspondence of SER excitation profiles to the surface plasmon frequencies of aggregates, and not to the dipolar plasmon frequencies of the single colloidal par- ticles, indicates that aggregation is essential to the observation of intense surface e n h a n ~ e m e n t . ~ ~ - ~ ~ Self aggregation may be observed on the adsorption of a number of analytes. However, for the majority of com- pounds, an aggregation procedure is required before SE- (R)RS can be observed. Various procedures have been used to induce adsorption and/or aggregation, the most common of which are addition of C1- or a mineral acid. The effect of C1- on the SERRS of dyes has been reported by Schneider et al., and the formation of aggregates was found to be dependent on the concentration of C1- added.I8 In addition, the procedure was found to be neither reproducible nor stable, and it was concluded that this greatly hindered quantitative analysis. Thus, in addition to the nature of the particle surface, the reproducibility and stability of the aggregation procedure is of importance if SE(R)RS is to be used for analytical purposes.

This study reports in more detail on the method of preparation of the citrate-reduced colloid and the surface properties of the colloidal particles. The stability and reproducibility of acid (HN03) aggregation with the citrate- reduced colloid is compared to an alternative procedure using poly(L-lysine) for the formation of stable and

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1992, 148, 129. (38) Xu, Y.; Zheng, Y. Anal.'Chim. Acta 1989,225, 227. (39) Xue, G.; Dmg, J.; Zhang, M. Appl. Spectrosc. 1991,45, 756. (40) Heard, S. M.; Grieser, F.; Barraclough, C. G.: Sanders, J. V. J . -

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Phys. B 1989, 49, 553.

2006.

3714 Langmuir, Vol. 11, No. 10, 1995

reproducible aggregates. The implications for qualitative and quantitative analysis by SE(R)RS are discussed.

Experimental Section Silver nitrate (99.9999%, Johnson Matthey), trisodium citrate,

ascorbic acid, sodium borohydride (Aldrich), poly(L-lysine) hy- drobromide (MW 4000-15 000, Sigma), Solvent Yellow 14, rhodamine 6G, pyridine, acetonedicarboxylic acid, acetoacetic acid, and formic acid were of analytical grade.

Citrate-reduced silver sols were prepared according to a modified Lee and Meisel method.31 All glassware (1000 mL round bottom flask, glass link stirrer) was rigorously cleaned before use by t reatment with aqua regia (HCl, HN03 (3: 1 v/v)) followed by gentle scrubbing in a soap solution and thorough rinsing with distilled water. A sample of silver nitrate (90 mg) was suspended in distilled water (500 mL, 45 "C) and heated rapidly to boiling under stirring ( the ra te of stirring was such tha t the solution vortex reached the base of the stirrer). Immediately, boiling commenced, a 1.0% solution of sodium citrate (10 mL) was added rapidly, and heating was reduced, but the solution was kept boiling gently for 90 min with continuous stirring.

Borohydride-reduced silver sols were prepared according to a modified Lee and Meisel method.31 All glassware was rigorously cleaned before use. An aliquot of aqueous silver ni t ra te solution (2.5 x M, 100 mL) was added dropwise to an ice water cooled aqueous sodium borohydride solution (2 x M, 300 mL). The resultant colloidal suspension was boiled for 1 h to remove excess borohydride and made up to 500 mL with distilled water.

For measurements by SERS using the borohydride-reduced col, individual solutions containing concentrations of approxi- mately M of each compound were prepared in distilled water. A sample of each solution (1OOpL) was added to separate aliquots of the borohydride sol (2 mL), and the colloidal mixtures were aggregated using dilute nitric acid (5 x lo-* M, 20-3OpL).

For SE(R)RS measurements using citrate-reduced colloid, individual solutions containing M analyte were prepared in distilled water (sodium citrate, pyridine, rhodamine 6G) or in 50% aqueous ethanol solution (Solvent Yellow 14, 2-naphthol). A sample of each analyte solution (100 pL) was added to separate aliquots ofthe silver colloid (2 mL). To promote aggregation, a n aqueous solution of poly(L-lysine) (0.01%, 150 pL) was added to the colloidal suspensions followed by an aqueous solution of ascorbic acid (1 M, 150 pL).

Visible absorption spectra in distilled water were recorded with a Perkin Elmer Lambda 16 Spectrophotometer using 1 m m quartz cuvettes. Raman spectra were recorded using a Spectra- Physics 2020 Argon ion laser (100 mW) as the excitation source (514.5 nm), with conventional 90" geometry. The spectra were recorded using a n Anaspec-modified Cary 81 scanning mono- chromator with a spectral resolution of 5 cm-l. A cooled Thorn EM1 9658R photomultiplier tube was used for detection, with photon-counting electronics for data acquisition. A microposi- tionable quadrant cell holder was employed for accurate and precise positioning of a 10 m m cuvette. Each spectrum took approximately 6 min to acquire. IH NMR spectra were acquired using a Bruker AMX 400 spectrometer a t 400.1 MHz, with samples being maintained at ambient temperature during acquisition.

Results and Discussions Optimization of Colloid Preparation. The proce-

dure reported by Lee and Meise131 for the formation of a colloidal silver SERS substrate by the reduction of silver nitrate with trisodium citrate was optimized to maximize the SERRS intensity of the azo dye Solvent Yellow 14. This required variation of a number of reaction conditions including the times and rates of reagent addition, heating, and stirring. The optimized procedure reported in the Experimental Section yields stable and approximately monodisperse colloids from which intense SERRS can be obtained when controlled aggregation is induced. (The aggregation procedure is discussed later.) The wavelength of maximum absorbance (A,,) is at approximately 402- 404 nm, which is consistent with the excitation of a dipolar

Munro et al.

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350 n 300 0

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Figure 1. Plots of the mean SERRS intensity for 10 replicate analyses of Solvent Yellow 14 (1235 em-') versus (a) fwhh and (b) A,,,=. (Error bars are shown.)

surface plasmon in silver spheres having radii of ap- proximately 10-20 nm in water.4s The full width at half- height (fwhh) of approximately 53-57 nm is consistent with moderate monodisperity within these colloids. The effects of deviation from the optimized procedure were examined. Minor bathochromic shifts of the Am= (1-2 nm), broadening of the absorbance bands (fwhh > 60 nm), and decreases in the absorbance at the A,, were observed for increases or decreases of the optimized stirring and heating rates and time and rate of reagent addition. Variation from the optimum rates for two or more parameters results in further bathochromic shifts (A,,, L 410 nm) and broadening of the absorbance bands (fwhh L 75 nm). These observations are consistent with the presence of an increasing number of linear aggregates andor prolate spheroid^.^^ Plot of the mean SERRS intensity for 10 replicate analyses of Solvent Yellow 14 (1235 cm-') versus fwhh and A,,, of the citrate-reduced colloid are illustrated in Figure 1. Adecrease was observed in the SERRS intensity with increases in both fwhh and A,,, and an increase in fwhh was observed with increasing Amax. However, for a given Amax, some variation in fwhh was noted. For example, for A,, = 405 nm, values of fwhh between 60 and 65 nm were obtained.

Visible Spectroscopy of Citrate Sol Particles. The reaction procedure under optimized conditions was char- acterized. Aliquots of solution withdrawn during colloid formation after 2, 4, 6, 8, 10, 15, 20, 30, 45, 60, 75, and 90 min were immediately cooled in an ice bath to prevent further progression of the reaction. The absorption spectrum was recorded for each aliquot. The Amax, corresponding absorbance, and fwhh values for aliquots withdrawn after 2, 10, 15, and 90 min are reported in Table 1, and their absorbance spectra are illustrated in Figure 2.

The A,, values for the aliquots withdrawn between 10 and 90 min were observed at approximately 404 nm (403 f 1 nm). The broad absorbance band (A,,, = 413 nm, fwhh = 89 nm) observed for the aliquot withdrawn after 2 min indicates that a polydisperse range of larger silver particles is initially formed in the suspension. This aliquot appeared yellow with transmitted light. A hypsochromic shift in Amax toward 404 nm was observed between 4 and 10 min. In addition, shoulders were observed at ap- proximately 380 and 430 nm and the bands broadened appropriately (fwhh = 122 nm). This resulted in a deep red appearance with transmitted light. These observa- tions indicate that the initial colloidal suspension re-orders and forms into smaller, more stable, and less polydisperse

(48) Kerker, M. J. Colloid Interface Sci. 1985, 105, 297.

Surface of a Citrate-Reduced Colloid Langmuir, Vol. 11, No. 10, 1995 3715

400 450 500 550

Wavelength (nm)

Figure 2. Visible absorbance spectra for aliquots withdrawn during colloid formation after (a) 2, (b) 10, (c) 15, and (d) 90 min.

Table 1. A,,, Absorbance, and fwhh for Aliquots Withdrawn during Colloid Formation after 2, 10, 15,

and 90 min time ( m i d Amax (nm) absorbance (A) fwhh (nm)

2 413 0.157 89 4 408 0.395 113 6 405 0.556 122 8 405 0.728 122 10 404 0.850 122 15 404 1.169 81 20 404 1.348 67 30 404 1.476 61 45 404 1.525 57 60 404 1.585 57 75 404 1.619 57 90 404 1.674 57

particles with time and are consistent with results on the formation of gold colloid by the citrate reduction of auric acid reported by Chow and Zukoski; large clusters are initially formed but dissociate into smaller particles as the reduction process continues.49

As previously mentioned, the band at 404 nm is consistent with spherical colloidal silver particles with radii of approximately 10-20 nm. The shoulders are indicative of coalescence of these particles, principally into doublets, but there may be some higher order linear aggregate^.^^ After 15 min, the intensity of the shoulders decreased dramatically: there was an accompanying decrease in the bandwidth (fwhh = 81 nm) and a sharp increase in the intensity of the absorbance at 404 nm. After 20 min no obvious shoulders were observed and the aliquots appeared yellow/green with transmitted light. The band continued to sharpen until a final, consistent fwhh of 57 nm (55 f 2 nm) was reached after 45 min. The absorbance at 404 nm increased with time. However, the rate of increase reduced toward 90 min, and after this time, the increase in absorbance is negligible. The visible absorbance spectra indicate that a heating time of 90 min is advised to produce a consistent particle size distribution and that the final colloid is close to monodisperse, consisting predominantly of single, relatively spherical silver particles with radii of approximately 10-20 nm.

Photon-Correlation Spectroscopy of Citrate Sol Particles. The average colloidal particle size for indi-

(49) Chow, M. K.; Zukoski, C. F. J . Colloid Interface Sci. 1994,165, 97.

Table 2. SER Frequencies for Aliquots Withdrawn during Colloid Formation after 2,10, 15, and 90 min.

The Values in Parentheses Are the Relative Intensities of Each Band with Respect to the Principal Band

(-1400 em-') SERS frequency (cm-l) at various time intervals

2 min 10 min 15 min 90 min

1390

1085 1025 952 933 903 840 796 760

336 232

1410 sh

1370 sh (1.00) 1395

(0.19) 1090 (0.29) 1025 (0.43) 953 (0.32) 934 (0.21) 903 (0.19) 840 (0.14) 800 (0.10) 760

(0.14) 336 (>1) 232

(0.91) (1.00) (0.77) (0.17) (0.39) (0.44) (0.33) (0.19) (0.20) (0.21) (0.11)

(0.70) (>I)

1410 sh (0.98) 1410 sh (0.95) 1400 (1.00) 1400 (1.00) 1370 sh (0.65) 1370 sh (0.63) 1090 (0.15) 1092 (0.14) 1025 (0.50) 1026 (0.48) 952 (0.27) 952 (0.28) 932 (0.18) 935 (0.22) 900 (0.12) 903 (0.11) 840 (0.11) 840 (0.17) 800 (0.23) 802 (0.26)

410 (0.17) 408 (0.14) 336 (0.46) 336 (0.40)

('1) 232 (0.98) 232

vidual aliquots withdrawn during silver colloid formation was determined using photon-correlation spectroscopy. The calculation used is 2-weighted. Therefore, size determinations were carried out for 3 and 10 ps sample times. A sample time of 10 ps is preferable for a mono- dispersed colloid, but a sample time of 3 ps reduces the weighting of the sample size toward the larger particles providing an improved estimation of the actual average particle size.

The size determinations correlate with the visible absorption data. The average particle size was determined to be approximately 20-30 nm, consistent with the silver particle size for which the dipolar surface plasmon excitation is observed at 404 nm. The average particle size for the aliquot withdrawn after 2 min was determined to be approximately 70-80 nm. In addition, aliquots withdrawn after 4 min were found to be highly polydis- perse (polydispersity L 1) with particles both in the 20- 30 nm range and the 40-50 nm range. However, the aliquot withdrawn after 90 min was found to be relatively monodisperse (polydispersity < 0.1) with an average particle size of approximately 27 nm.

The conclusions of the absorption and photon-correlation spectroscopic studies are further strengthened by previous studies by electron microscopy of colloids evaporated on a glass slide. The particles appear to be multifaceted crystallites with a somewhat hexagonal structure, but the dimensions are relatively uniform, and they ap- proximate to spheres with diameters of 24-30 nmq50

SERS Examinationof the Citrate-Reduced Colloid Surface Species. The species a t the surface of the colloidal particles formed during reduction by the citrate method were examined by SERS. Aliquots (2 mL) were withdrawn at timed intervals during the formation of the silver colloid. Prior to aggregation, only background scattering was observed. However, on aggregation by the addition of dilute nitric acid (5 x M), intense SERS from the species a t the silver surface was observed. The frequencies of the SER bands for the aliquots withdrawn after 2, 10, 15, and 90 min are listed in Table 2, together with the relative intensities of each band with respect to the principal band (-1400 cm-'1. The corresponding spectra are illustrated in Figure 3.

After 2 min, the principal band was at 1390 cm-l and the next most intense band at 952 cm-l. After 10 min, the principal band had shifted to approximately 1395 cm-l and had intense shoulders a t approximately 1412 and

(50) Rodger, C.; Edmondson, M.; Dent, G. Unpublished results.

3716 Langmuir, Vol. 11, No. 10, 1995 Munro et al.

i \ X3 i

1600 1200 800 400

Wavenumbers (cm-1)

Figure 3. SERS from aliquots withdrawn during colloid formation after (a) 2, (b) 10, (c) 15, and (d) 90 min.

1370 cm-'. No notable changes in frequency and relative intensity were observed for the other bands, with the exception of the band at 1025 cm-' which increased in relative intensity by approximately 30%. After 15 min, a number of notable spectral changes were recorded. In addition to a further shift in frequency for the principal band to 1400 cm-l, the band at 760 cm-' was no longer observed and a new band was observed at approximately 410 cm-l. Significant changes in relative intensity were also observed. In particular, a further increase in intensity was observed for the band at 1025 cm-'. This was accompanied by a decrease in relative intensity for many other bands, including those observed at 952, 932, and 903 cm-'. Few large changes were noted for aliquots withdrawn between 15 and 90 minutes. However, a 10- fold increase in the overall scattering intensity was observed. (It should be noted that this is only an approximate value as the state of aggregation induced may not have been optimum for each sample examined.)

This examination confirms the presence of an organic layer a t the colloid surface and indicates the presence of a t least two surface species during colloid formation. NMR Examination of Solution Species Present

during Reduction by Citrate. The solution species present during reduction by citrate were examined by lH NMR spectroscopy. Aliquots of solution were withdrawn during colloid formation after 1, 1.5, 2.5, 3.5, 5, 7, 10, 15, 30,45,60, and 75 min and were immediately cooled in an ice bath to prevent further progression of the reaction. A portion (0.45 mL) of each aliquot was placed in an NMR tube containing D20 (0.05 mL), and the lH NMR spectra

Figure 4. NMR spectra for aliquots withdrawn during colloid formation after (a) 1.5, (b) 3.5, (c) 7, and (d) 60 min.

were acquired using the P133i sequence with a 90" pulse of 26 This sequence removes the residual H20HDO signal by selectively exciting the areas to either side of the dominant H20HDO signal.

The spectra recorded for aliquots withdrawn after 1.5, 3.5, 7, and 60 min are illustrated in Figure 4. These indicate clearly the change in concentration and nature of the organic species in solution during preparation of the colloid. Immediately after the addition of citrate to the silver nitrate solution, only sodium citrate was detected. However, after 3.5 min additional peaks were observed at 2.27 and 3.51 ppm, consistent with the formation of the silver salt of acetoacetic acid in solution. A further small peak observed at 8.44 ppm corresponds to formate.

After 7 min, additional peaks were observed at 2.22 and 3.43 ppm, indicating the presence of acetoacetic acid. After 10 min, the concentrations of the silver salt of acetoacetic acid and of acetoacetic acid were approximately equ_al. (Due to the nonuniform excitation pattern of the P1331 sequence the integrals of peaks at different frequencies cannot be compared; however, in this case, as the peaks are close in frequency, the signal to noise ratio probably contributes more to the error in area than the nonuniform excitation profile.) After 60 min almost no citrate was observed in solution and the concentration of free acetoacetic acid was approximately 3 times that of the silver salt. These observations are consistent with the thermal decomposition of citric acid (I). The intermediate product, acetonedicarboxylic acid (II),

(51) Hore, P. J. J. Magn. Reson. 1983, 55, 539

Surface of a Citrate-Reduced Colloid Langmuir, Vol. 11, No. 10, 1995 3717

Table 3. SER Frequencies for Citrate and Its Decomposition Products, Acetonedicarboxylic Acid and

Acetoacetic Acid, at the Surface of Borohydride-Reduced

Intensities of Each Band with Respect to the Principal Band

SERS frequency (cm-l)

Colloid. The Values in Parentheses Are the Relative

was not observed in the solution as it is highly unstable at elevated temperature and spontaneously decarboxy- lates to form acetoacetic acid (111).

SERS Using Borohydride-Reduced Silver Colloids of the Solution Species Identified by NMR. The reduction of silver using borohydride produces a colloid with particles close in size (10- 15 nm) to those produced by the citrate method, but aggregation with nitric acid results only in the observation of Raman scattering from water, indicating that no organic SERS-active species are adsorbed at the silver surface. Thus, the adsorption of solution species directly onto this surface may provide a good model on which to base an understanding of the surface layer resulting from citrate reduction. Each of the species indicated by the NMR study, i.e., citrate and its decomposition products, acetoacetic acid and formate, were added to the borohydride-reduced colloid, and aggregation was induced with nitric acid. In addition, acetonedicarboxylic acid and acetone were examined as these are intermediate and final products in the decom- position pathway of citrate and, although not identified as being present by NMR, it is possible that these products are absorbed and stabilized at the surface. Self aggrega- tion, but not SERS, was observed on the addition offormate to the borohydride colloid. A possible explanation is that the freshly reduced silver surface is reoxidized in the aqueous solution by 0 2 , and on addition of formate, complexation with silver(1) takes place. However, the complexes readily dissociate from the surface, with etching of the surface as is the case for aluminum and oxalic acid, and chromium and amino acids.52 Self aggregation was not observed with the other species. On aggregation with nitric acid, no SERS was observed from acetone and poor SERS, which was barely distinguishable from the water signal, was observed from acetoacetic acid. However, intense SERS was recorded for citrate and acetonedicar- boxylic acid. The frequencies of the observed bands are listed in Table 3. The relative intensity of each band with respect to the principal band (-1400 cm-'1 is reported in parentheses for citrate and acetonedicarboxylic acid but not for acetoacetic acid due to the dominance of the water signal. Citrate, acetonedicarboxylic acid, and acetoacetic acid all exhibit an intense SER band at approximately 1390-1400 cm-l. Marked differences were noted in the SERS from each species, enabling discrimination of the different adsorbates at the surface.

Determination of the Surface Species of the Citrate-Reduced Silver Colloid. Comparison of the SERS for the aliquots of citrate-reduced colloid with those for the solution species examined on the surface of a borohydride-reduced sol reveals similarities with adsorbed citrate but not with any other solution species. The SER frequencies and relative intensities of bands for citrate on the borohydride sol compare directly with those for the surface species after 2 min but not with those after 90 min. Additional citrate was added to an aliquot of silver colloid withdrawn after 90 min. The SER frequencies and relative intensity of each band were found to almost exactly match those for the aliquot of citrate-reduced

(52) Brown, D. H.; Smith, W. E.; Fox, P.; Sturrock, R. D. Inorg. Chim. Acta 1982, 67, 27.

sodium acetonedicarboxylic acetoacetic citrate acid acid

1390 (1.00) 1708 (0.45) 1648 1085 (0.10) 1618 (0.56) 1576 1024 . (0.21) 1588 (0.56) 1517 955 (0.43) 1551 (1.00) 1442 933 (0.21) 1410sh (0.32) 1400 903 (0.12) 1388 (0.61) 1337 839 (0.12) 1360 sh (0.48) 1265 798 (0.15) 1302 (0.28) 1190 763 (0.16) 1221 (0.36) 1085 338 (0.37) 1167 (0.26) 935

1104 (0.47) 812 980 (0.18) 616 958 (0.17) 933 (0.20) 900 (0.08) 824 (0.04) 760 (0.06) 695 (0.13) 630 (0.17)

Table 4. Raman Frequencies and Assignments for Aqueous Trisodium Citrate (1.5 M). The Values in

Parentheses Are the Relative Intensities of Each Band with Reqpect to the Principal Band (-1417 cm-9

frequency (cm-9 assignment frequency (cm-1) assignment 1575 1417 1300 1267 1212 1146 1095 1057 956 9 12

(0.15) (1.00) (0.11) (0.09) (0.09) (0.08) (0.20) (0.15) (0.58) (0.15)

v,(COO) 843 v,(COO) 812

762 d(C00) 670 6(COO) 622

596 560

v(C-0) 408 v(C-COO) 325 V(C -COO)

(0.44) v,(CCCC-O) (0.16) (0.13) (0.15) 6(COO) (0.21) (0.21) 6(COO) (0.23) 6(COO) (0.29) @(COO) (0.24)

colloid withdrawn after 2 min and thus also corresponded to those observed for citrate on the borohydride-reduced sol.

These results indicate that citrate forms a surface layer on the citrate-reduced colloid. However, the differences between the SER spectra indicate that the final citrate layer is oriented andor bound differently to excess citrate adsorbed onto the surface. To investigate this further, the SER spectra were compared with the ordinary Raman spectrum of aqueous sodium citrate (1.5 MI. The Raman frequency and relative intensity of each band together with tentative assignments of a number of citrate bands are reported in Table 4.

The carboxylate symmetric stretching mode, v,(COO), in the Raman spectrum from trisodium citrate in solution consisted of a single narrow band at 1417 cm-l. This band is broadened and shifted to approximately 1390 cm-l in the SER spectrum from the aliquot withdrawn after 2 min, indicating interactions between the carboxylates and the silver surface. It is intense, indicating that the Y,- (COO) modes have components perpendicular to the surface.53 This is supported by the strong intensity of the bands at 952,933, and 903 cm-l which are assigned to the three carbon-carbon stretching modes, v(C-COO), and also the disappearance of the bands at 1267, 1212, and 670 cm-l which are associated with the carboxylate

~~

(53) Creighton, J. A. In Spectroscopy of Surfaces; Clark, R. J . H., Hester, R. E., Eds.; John Wiley: Chichester, 1988; p 37.

3718 Langmuir, Vol. 11, No. 10, 1995

deformations, &COO). A strong increase in relative intensity, with respect to the solution spectrum, is also observed for the band at 1025 cm-l. This band has been tentatively assigned to the carbon-oxygen stretch, Y(C- 0), of the tertiary alcohol. Furthermore, two additional bands were observed in the SER spectra a t 232 and 336 cm-l. The intense band at 232 cm-l is assigned to the silver-oxygen mode, (COO-Ag). This is dominant after 2-15 min, but it should be noted that no correction is made for the encroachment of background Rayleigh scattering a t this frequency. The effect this will have on the measured intensity will be less noticeable after 90 min than after 2-15 min due to the 10-fold increase in overall Raman intensity with time. The band at 336 cm-' is consistent with the pseudocycle mode which is observed in succinate.54 This mode is only observed if the two carboxylates of succinate are eclipsed. A similar mode may be mimicked in the citrate ion by eclipsing of the tertiary carboxylate with one of the terminal carboxylates.

There is a rearrangement of the citrate ion at the surface after 15-90 min of heating, as indicated by the observed spectral changes discussed previously. The shift of the principal v,(COO) band to 1400 cm-l and the appearance ofshoulders a t approximately 1412 and 1370 cm-' indicate the interaction of the three carboxylates with the surface to be nonequivalent. The shoulder a t 1412 cm-' can be tentatively assigned to an unbound carboxylate and the bands at 1400 and 1370 cm-l to two nonequivalent bound carboxylates. This can be best rationalized by the binding of one terminal carboxylate and the tertiary carboxylate to the silver while the second terminal carboxylate remains unbound. The shift to lower energy observed for the two bound groups is consistent with monodentate binding to the metal. The high relative intensity of each carboxylate band would indicate that all of these modes still have components perpendicular to the surface. The three Y- (C-COO) modes remain intense. However, the increase in intensity observed is not as strong as that for the Y,- (COO) and Y(C-0) modes. In particular, the further increase in relative intensity of the Y ( C - 0 ) mode indicates that it has reoriented increasingly perpendicular to the surface. The surface layer could be postulated as consist- ing ofpolymeric silver citrate.55 However, such compounds are complex and involve binding via all three carboxylate groups. The picture which emerges is of a well-organized monolayer formed after 90 min of heating which, on addition of excess citrate, is disrupted by the formation of a less organized, thicker layer of citrate involving hydrogen-bonding interactions. A model of interaction and orientation of the organized monolayer a t the surface after 90 min, which best rationalizes the spectral infor- mation, is proposed in Figure 5. This model is based on the interaction of a single citrate molecule with the silver surface. Intermolecular hydrogen bonding may occur via the hydroxyl of the tertiary alcohol group. Furthermore, the nature of the silver oxide surface is not known and, therefore, a simplified flat silver surface is illustrated for the purpose of this model. The geometry of the adsorbed citrate molecule is in close agreement with the optimized geometry of the trisodium salt in solution.56

Colloid Aggregation Procedure. Aliquots of colloid (2 mL) and Solvent Yellow 14 (lo+ M, 100 pL) were aggregated by the addition of different volumes of nitric acid (5 x M, 0-120pL), and the intensity of SERRS (1235 cm-l) was recorded. Ten replicate analyses were

Munro et al.

~ ~~~

(54) Moskovits, M.; Suh, J. S. J . A m . Chem. SOC. 1985, 107, 6826. (55) Sagatys, D. S.; Smith, G.; Bott, R. C.; Lynch, D. E.; Kennard,

(56) Tarakeshwar, P.; Manogaran, S. Spectrochzm. Acta 1994,50A, C. H. L. Polyhedron 1993, 12, 709.

2327.

c-c

i ~ - A3 Ag- Figure 5. Proposed model of interaction and orientation of cit;ate at tk colloid surface.

0 30 60 90 120

Volume H N S ( p I) I Figure 6. Plot of mean SERRS intensity for 10 replicate analyses of Solvent Yellow 14 (1235 cm-') versus volume of HNO3 (5 x M: 0-120 pL) added to citrate colloids with fwhh = 55,65, and 75 nm.

carried out for each volume of acid added. This was repeated for two nonideal colloids (Amm = 404 nm, fwhh = 65 nm and Amax = 410 nm, fwhh = 75 nm). A plot of average SERRS intensity vs volume of HN03 (5 x M) added, for each colloid, is illustrated in Figure 6. The scattering enhancement increases with the volume of acid added until maximum scattering intensity is observed, after which the colloid destabilizes and begins to pre- cipitate, and the observed signal decreases accordingly. The volume of acid required for optimum SERRS en- hancement from an ideal colloid (Amm = 402 nm, fwhh = 55 nm) was 90 pL, and the relative standard deviation (RSD) was 18.5%. The absorbance spectra of the colloidal mixtures have a broad shoulder to the red of the 402-404 nm band indicating polydisperse aggregates (Figure 7). The strong absorbance at 402-404 nm indicates that approximately 55% of the colloidal particles are unag- gregated.

The volumes of acid required for optimum scattering enhancement and the RSDs for the nonideal colloids were found to be 60 pL, 25.0% (A,,, = 404 nm, fwhh = 65 nm) and 30 pL, 16.2% (Amm = 410 nm, fwhh = 75 nm). The aggregation procedure has a clear dependence on the quality of the citrate-reduced colloid. For example, 60 p L is insufficient to produce optimum scattering enhancement from a well-prepared colloid (Amm = 402 nm, fwhh = 55 nm). However, the same amount would destabilize a less ideal colloid (Amm = 410 nm, fwhh = 75 nm).

A high degree of reproducibility (RSD '5%) is a general requirement of a good analytical technique. However,

Surface of a Citrate-Reduced Colloid Langmuir, Vol. 11, No. 10, 1995 3719

-..- ......... _.___- -___.____.__.__ .--.__ I I I I I I I

400 500 600 700

Wavelength (nm)

Figure 7. Visible absorbance spectra for a citrate colloid aggregated with (a) HN03 and (b) poly(L-lysine) and ascorbic acid. (The dotted line represents the spectrum for citrate colloid prior to aggregation.)

under optimum conditions the reproducibility is very poor (RSD = 18.5%), and this, as with the Cl--based proce- dure,18 hinders quantitative SERRS analysis. In addition, the instability reported with the C1- procedure was also noted with HN03 aggregation. The maximum scattering (55 nm fwhh colloid, 9OpL HN03) was found to maximize approximately 9 min after the addition of the acid and then to decrease at an approximate rate of 30%/h. Therefore, the band intensities of spectra acquired during scanning or data accumulation will be altered and will depend on the scan rate or accumulation time.

M, 100 pL) were aggregated by the addition of poly(L- lysine) (0.01% d v , 150 pL) and ascorbic acid (1 M, 150 pL), and the SERRS intensity (1235 cm-l) was recorded for 10 replicate analyses. The use of this procedure to promote adsorption of anionic dyes to and aggregation of a citrate-reduced colloid was demonstrated p rev iou~ ly .~~ The protonated amino groups of the lysine residues of poly(L-lysine) are attracted to and readily adsorbed onto the negatively charged citrate layer at the surface of the sol particles leading to controlled aggregation of the particles and an intense SERRS effect. The quantity of ascorbic acid used does not induce appreciable aggregation in the silver colloid alone, and no significant surface enhancement is observed. In conjunction with poly(L- lysine), it promotes the protonation of the lysine residues, increasing the efficiency of the process. The scattering enhancement approximately doubles with respect to that for optimum acid aggregation. In addition, both the reproducibility and the stability of the aggregates were significantly improved. The reproducibility (RSD = 4.6%) indicates that this method may improve quantitative analyses by SERRS. There was no observable change in scattering intensity after 1 h enabling effective evaluation of band intensities.

The visible spectra indicate notable differences between the aggregates formed with poly(L-lysine) and ascorbic acid and those formed with nitric acid (Figure 7). In place of the broad, red-shifted shoulder, a second distinct maximum (650-700 nm) was observed. In addition, only approximately 40% of the colloidal particles remain unaggregated, compared with 55% with optimum nitric acid aggregation. Aggregation is not only more reproduc- ible and stable but also more complete and less polydis- perse.

Aliquots of colloid (2 mL) and Solvent Yellow 14

Ramen Intasm

Ll

1600 1200 800 400

Wavenumbers (cm-1)

Figure 8. SE(R)RS from submonolayer concentrations of 2-naphthol and Solvent Yellow 14 (a and b, respectively) on aggregation with HN03 and (c and d, respectively) on aggrega- tion with poly(L-lysine) and ascorbic acid.

A notable feature of aggregation of the citrate-reduced colloid with poly(L-lysine) and ascorbic acid in the absence of analyte is that, unlike aggregation with nitric acid, no SERS is observed from the citrate layer. Furthermore, no signals are observed from poly(L-lysine), ascorbate, or water.

The effectiveness of the poly(L-lysine) and ascorbic acid method with different analytes was examined. The view of the colloid which emerges is of a silver particle with an organized monolayer of citrate bound to the surface, presumably to a thin silver(1) oxide layer, with pendant negatively charged groups. Analytes adsorbed at the colloid surface could replace citrate and complex with silver(1) (pyridine), form bonds with the negatively charged pendant groups (rhodamine 6G), or physically adsorb at the citrate layer (2-naphthol, Solvent Yellow 14). To examine the effect of each type of adsorption, low concentrations of pyridine, rhodamine 6G, 2-naphthol, and Solvent Yellow 14 were adsorbed individually a t the colloidal surface, such that aggregation with nitric acid yielded SE(R)RS from both the analyte and the citrate surface layer. Aggregation with poly(L-lysine) and ascorbic acid resulted in intense SE(R)RS from each analyte and a slight increase in the overall intensity of the analyte. However, no SERS was observed from the citrate layer. The SE(R)RS from 2-naphthol and Solvent Yellow 14 on aggregation of the colloid with nitric acid and with poly- (L-lysine) and ascorbic acid are illustrated in Figure 8. Solvent Yellow 14 and pyridine were co-adsorbed at the surface such that aggregation with nitric acid yielded SE-

3720 Langmuir, Vol. 11, No. 10, 1995

(R)RS from both analytes. On the addition ofpoly(L-lysine) and ascorbic acid the overall SE(R)RS intensities of both analyte signals increased with respect to nitric acid aggregation, but there were no differences in the relative contribution of each signal. The use of poly(L-lysine) and ascorbic acid is shown to be equally effective in the analysis of submonolayer concentrations of neutral and cationic SERS- and SERRS-active species chemisorbed or phys- isorbed at the colloid surface, in addition to the anionic SERRS-active species previously examined.14

The differences in aggregation procedures require further examination to explain the effect on SERS from the citrate surface layer. However, the advantages of the poly(L-lysine) and ascorbic acid procedure for SERS and SERRS analyses are clear: the method is robust, the reproducibility of SE(R)RS (RSD '5%) is acceptable for analytical purposes, and submonolayer analyte mixtures can be examined without interference from the stabilizing citrate surface layer.

Colloid Stability. Colloids produced by the reported method are stable with time. No changes are observed in the visible absorption spectra, or SERS from the citrate surface layer, for colloids examined 2 months after preparation. SE(R)RS intensities from analytes adsorbed on aged sols are the same as those from freshly prepared sols within experimental error.

Conclusions The intensity of surface-enhanced (resonance) Raman

scattering from an analyte adsorbed at the surface of a citrate-reduced silver colloid is dependent on the reduction procedure. Colloids prepared under the optimum condi- tions described have a wavelength of maximum absor- bance (Ama) at approximately 402-404 nm and afull width at half-height (fwhh) of approximately 53-57 nm. Devia- tions from the optimum rates of heating and stirring and the time and rate of reagent addition result in batho- chromic shifts and broadening of the absorbance spectra (A,,, I 405 nm, fwhh I 60 nm). Such increases in A,,, and/or fwhh are accompanied by a decrease in the intensity of surface-enhanced (resonance) Raman scattering and the stability of the aggregate. Thus visible absorption spectra can thus provide a simple means of quality control in the production of a SE(R)RS substrate.

Visible absorption and photon-correlation spectroscopic studies of the colloid formation process indicate that the initial reduction ofAg' to AgO occurs within 2 min of citrate addition and that initial particles formed are large (60- 80 nm) and polydisperse. Subsequent treatment initially

Munro et al.

provides a less polydisperse mixture (20-30 nm and 40- 50 nm particles) and finally an approximately monodis- perse distribution ofthe smaller particles (-27 nm). NMR studies indicate the presence of citrate and its decomposi- tion products, acetoacetic acid and formate, throughout colloid formation. Raman scattering indicates that two forms of citrate but no decomposition products are adsorbed at the silver surface. The final, approximately monodisperse, particles are believed to be stabilized by an organized surface monolayer of silver citrate, with pendant negative groups. The colloids are stable for a t least 2 months, and each preparation provides suficient substrate for approximately 250 SE(R)RS analyses (based on 2 mL colloid per sample).

The effectiveness of poly(L-lysine) and ascorbic acid for the manipulation of the negatively charged citrate surface layer to enhance adhesion of anionic SERRS-active species and to induce aggregation has been illustrated previ~usly.~ This method is shown to be equally effective in the analysis of neutral and cationic SERS- and SERRS-active species chemisorbed or physisorbed at the colloid surface. Ag- gregation with poly(L-lysine) and ascorbic acid is more effective than that with acid (HN03). It produces more monodisperse colloidal aggregates, and the reproducibility is improved to an extent which is acceptable for analytical purposes (RSD < 5%). Furthermore, in addition to an increase in SE(R)RS from the analyte, no interfering signals are observed from the citrate surface layer. This effect requires further study, but it is an advantage for trace analysis.

From a practicalviewpoint, this study provides sufficient definitions and suggests simple guidelines for the produc- tion and quality control of the citrate-reduced colloid to enable reproducible colloid production by the citrate method in all laboratories. It also provides a robust method for reproducible SE(R)RS analysis which has been employed for sensitive and selective qualitative and quantitative analysis. In addition, through the under- standing and manipulation of the colloid surface, it provides a starting point for the development of new analytical procedures which could unlock the potential of SE(R)RS for routine trace analysis.

Acknowledgment. We thank Dr. Whatley of the Pharmacy Department a t Strathclyde for his help in obtaining photon-correlation measurements and EPSRC and Ciba Pigments, Paisley, for financial assistance.

LA950158A