Anal. Chem. 1995, 67, 3187-3192
Detection of Aromatics in Aqueous Solution by SurfacemEnhanced Raman Scattering by Substrates Chemically Modified with p-tert-Butylcalix[4]arenetetrathiol Wieland Hill,**t Bemhard Wehling,t Charles 0. Gibbs,* C. David Gutsche,* and Dieter Kluckowt lnstitut f i r Spektrochemie und Angewandte Spektroskopie (ISAS), P.O.Box 101352,44013 Dortmund, Germany, and Texas Christian University, P.0. Box 32908, Fort Wotth, Texas 76129
Chemical modificationof rough silver surfaces byp-tertbutylcalix[4larenetetrathiol (BCAT) has been demonstrated to produce substrates for surface-enhanced Raman scattering (SERS)that form reversibly complexes with aromatics from aqueous solutions. Due to this complexation, the detection limits for aromatics without groups that attach to silver are clearly decreased in comparisonto mere adsorption at the bare metal surface and were found to be 100 pM for benzene, 50 pM for chlorobenzene, and 5 pM for l,%-dichlorobenzenein water. The SERS bands of the surface-boundBCAT have been used as an internal standard for the surface concentration of aromatics. The intensity ratio of bands of adsorbed aromatics and surface-boundBCAT has given a measure for the solution concentration of the aromatics with a dynamic range of about 2 orders of magnitude. Characteristic shifts of aromatics bands due to substitution have permitted the identifkation of benzene derivatives as well as mixhve analyses. An adsorption enthalpy of -6.7 kJ/mol has been determined for the adsorption of chlorobenzene by measuring its temperature dependence. Recent improvements of instrumentation clearly extend the fields of application of Raman spectroscopy using compact, sensitive, and portable spectrometers. The surface-enhanced Raman scattering (SERS) at rough metal films or particles is a rather promising tool for chemical sensors because of its ability to easily detect submonomolecular layers of organic substances adsorbed at metal surfaces. A major problem in the application of SERS to chemical sensing arises from the necessity for adsorption at a metal surface. Metals exhibit rather unspecific adsorption properties and are chemically reactive. Therefore, analytes may not be adsorbed or adsorption centers may be poisoned by concomitant substances. Such problems can be avoided by organic modification of SERS active metal substrates. By chemical modification, selectively adsorbing centers can be generated. However, at a modified surface, analytes interact with the organic adsorption centers and not directly with the metal surface. Therefore, the chemical contribution to the SERS ISAS. :Texas Christian University.
(1)McGlashen. M. L.;Davis. K. L.; Morris, M. D.Anul. Chem. 1990,62,846849. 0003-2700/95/0367-3187$9.00/0 0 1995 American Chemical Society
enhancement due to bonding to the metalz may be suppressed for the analyte. Chemically enhanced SERS bands of the mo&ed layer might superpose the weaker analyte bands. We will show that this effect does not dominate for the investigated system. Furthermore, iodide ions that were shown in a previous pape6 to exhibit selective enhancement proved to be useful for a selective enlargement of analyte bands in comparison with those of the modified layer. Thioorganics such as thiols or disulfides are known to be useful agents for the organic modification of metal surfaces, since the sulfur functions react chemically with the metal, yielding stable surface compound^.^ Adsorption of chlorinated ethylenes and of aromatics at SERS substrates hydrophobized with octadecanethiol has been demonstrated in recent paper^.^^^ Lipophilic interaction resulted in a concentration of the investigated organics within the SERS active layer and lowered the SERS detection limits. A first example of stoichiometricadsorption at thioorganically modified SERS substrates was the complexation of alkali metal ions with immobilized thiol-derivatized diben~o-l&rown-6.~Recently, SERS was used to detect complexation of methyl orange at colloidal silver coated with thiol-derivatized cyclodextrin.* In the present paper, evidence for reversible complexation of organic molecules with organically modified SERS substrates is reported. Since organic analytes in general exhibit characteristic SERS bands, a double selectivity can be achieved by this detection principle: selective complexation combined with specific detection. EXPERIMENTAL SECTION Raman Spectroscopy. Raman measurements were carried out with a 0.5 m triple monochromator with subtractive dispersion of the first two stages OILOR XY). The dispersion of the spectrometer was 1.1nm/mm. The excitation source was a cw Ti:sapphire laser (Coherent 890) operating at a wavelength of 702 nm. The laser radiation was filtered by a grating monochromator (2) Otto, A.; Mrozek, I.; Grabhom, H.; Akemann, W. J. Phys.: Condens. Mutter
1992,4,1143-1212. (3)Wehling, B.; Hill, W.; Klockow, D. J. Mol. Stmct. 1995,349,117-120. (4) Ulman, A. An Introduction to Ultrathin Organic Films, From LangmuirBlodgett to SevAssembly; Academic Press: Boston, 1991. (5) Mullen, K.; Carron, K. Anal. Chem. 1994,66, 478-483. (6) Carron, IC;Peitersen, L.; Lewis,M. Enuiron. Sci. Technol. 1992,26.19501954. (7) Heyns, J. B.; Sears, L. M.; Corcoran R C.; Carron, K. T.Anal. Chem. 1994, 66,1572-1574. (8) Maeda, Y.;Kitano. H. J Phys. Chem. 1995,99, 487-488.
Analytical Chemistry, Vol. 67, No. 18, September 15, 1995 3187
laser
\
-
EoIleaion optics
f
.
dumina ~ o l c s slide
Figure i.Schematic of a SERS substrate.
to remove lowintensity sidebands and the spontaneousemission background. Laser light with a power of -10 mW was focused to the sample on a spot with a diameter of 30 pm. An f/l.O collection optics produced a Sfold enlarged intermediate image of the laser spot in the plane of the entrance slit of the spectrometer. Slit widths were 300 pm. A nitrogencooled CCD camera (Wright Instruments) with a 298 x 1152 chip (EEV88131) sewed as the detector. Integration times were 5 min, unless otherwise stated. SERS spectra were taken in backscattering geomehy from substrates F i r e 1) immersed in aqueous solutions of aromatics contained in a conventional 1an quartz cuvette for fluorescence measurements. 'The cuvetteswere closed to prevent evaporation of volatile analytes during the measure ments. 'The analyte solution was thermostated by a water thermostat connected to a homemade cuvette holder within a temperature range from 274 to 343 K Peak heights of SERS bands of the analytes were determined using difference spectra of subshates immersed in analyte solution and in pure water. ?he given detection limits are based on measurements with analyte peak heights of minimum 3 times the standard deviation of the difference spectra background. Materials. Rough metal films of a thickness of 75 nm were prepared by vacuum evaporation of 99.99% silver onto glass slides previously s p k o a t e d with 15OpL of aqueous suspensions of 0.3 pm alumina particles accordii to a method described by Bello et al.9 Scanning electron microscopy showed that the substrates exhibit submicrometer roughness due to separate alumina particles as well as larger structures of aggregates. fifni-sutyl~[4larenetetrathiol@CAT) was prqxred from fif&-butylcalix[4larene (BCA) as described elsewhere.10 Siiver SERS substrates were coated with BCAT by immersion of the subshates for 90 min in 100pM BCAT solutions in toluene. 'The dependence of SERS intensities on the immersion time indicated practically completed adsorption of BCAT after 90 min. In the standard procedure, BCATcoated substrates were immersed for 5 min in 1mM aqueous KI solutions and rinsed with highpurity water before the adsorption experiments with aromatics, Adsorp tion of iodide ions is known to enhance the SERS intensities by chemical effects."J* m e iodide was not applied for measurements of the subsection "complexation". (9) Bello. I. M.;Stokes. D. L:VeDinh. T.AppL Spa-imz, 1989.43.13251330. (10) Gibbs. C. G.; Gutsche. C. D.J A m atem. S a 1993, 215.5338-5339. (11) leanmaire. D. L: Van Dupe. R P. J. Elechonnol am.1977.84.1-20. (12) Pettinger. B.: W e 4 H.In Srrfnrc Exkmced Ram%hrminp: Chang. R K. Fumk. T.E., Eds.; Plenum Res: New York and London. 1982 pp 293-314.
3188 Anamal Chemistry. Vol. 67, No. 18, September 15, 1995
Benzene &le& >99.5%GC),chlorobenzene &le& >99.5% GC), 1,Zdichlorobenzene (Fluka, >99% GO,hexachlorobenzene (Schuchardt, >9a), and toluene Werck, >99.7% uvasol) were used as delivered for the preparation of aqueous and toluenic solutions. High-purilywater with an electrical conductivity below 0.07 pS was produced by a commercial water purification system (Millipore MiUIQ) equipped with an additional filter for O~aIliCS Concentrated aqueous solutions of the analytes (20 mM benzene, 2 mM chlorobenzene, and 0.5 mM l&Iichlorobemne, respectively) were produced by pipetting with a newly calibrated pipet the desired amount of analyte and mixing it with the solvent within a closed, calibrated glass bulb. Complete dissolution of chlorohenzenes was achieved by ulhasonic @ament. Diluted solutions were obtained by pipetting from the concentrated original solutions immediately before the SERS measurements. 'The saturated aqueous solution of hexachlorobenzene was prcduced by lasting treatment of a mixture of the analyte with water in an ultrasonic bath. safety Considerations. 'The analytes used here are toxic and/or carcinogenic. Swallowing, skin contact, and breathing in of these chemicals should be shictly avoided. Intense collimated laser light can cause skin irritation. Observation of collimated or scattered laser light with unprotected eyes can lead to serious eye damage. RESULTS AND DISCUSSION Immobilization. Basket-shaped ealixarenes are known as complex-forming agentsfor various compounds.1"4 Replacement of the hydroxyl groups of the lower rim of BCA by thiol groups1o produces an agent that can be easily immobilized at metal surfaces by the procedure described above. However, the strong inhmclecular hydrogen bonding among the OH groups is assumed to conhibute essentially to the formation of the cone conformation F i r e 2, structure l),in which BCA exists almost exclusively in solution and in the solid state.'O 'The thiol groups exhibit weaker hydrogen bonding, and the BCAT was found to exist in the 1,Mternate conformation (Figure 2, structure 2) in both the solid and the solution state.'O Complexing capabilities of immobilized BCAT should shangly depend on its conformation. merefore, the question arises whether the cone conformation can be restored by bonding to the surface or the alternate conformation predominates there also. A comparison of the SERS spectrum of the immobilized BCAT with the Raman spechum of the same suhstance in the solid form before immobilization ( F i i 3) shows a complete disappearance of the S-H stretching bands around 2540 cm-l after bonding to the surface. 'Thus. it can be assumed that at least the major part of the sufacehund molecules has formed four S-Ag bonds with the surface. Since this 4fold bonding is hardly imaginable for the alternate conformation, one should a s s m e that the conforma tion of the BCATchanged to conelike ( F i i e 2, structure 3)due to the bonding to the surface. ?his assumption is further supported by the complmhg capabiies of the immobilized BCAT described below. Further differences between the spefha of the immobilized BCAT and the solid reagent may also be caused by the change of conformation. However, a detailed analysis of the (13) Gutsche, C. D. C a l i e s . In Maosmpkr in Supmmoblar Chmist~~, Stoddart, J. E. Ed.; Royal Society of Chemishy: London. 1989. (14) B6hmer. V.. Vieens. J.. Eda Colknnrr. A V e ~ t i l C?as e ofMo-lic Compmim; Kluwer Academic Publishers: Dordrecht 1991.
2
1
3
Figure 2. Conformation of caiix[4]arene ( l ) ,its thio derivative BCAT (2), and the surface-bound molecule 2 (3).
Wavekngth Sbift (om-1) Figure 3. SERS spectrum of ptert-butylcalix[4]arenetetrathiol after bonding to a silver substrate (a) and Raman spectrum of the same calix[4]arene in the solid state (b). (Bands in spectrum a are stronger than those in the following figures due to the use of a stronger enhancing SERS substrate charge.)
vibration modes of this comparatively large molecule cannot be given at present. Complexation. Benzene is lacking functional groups that can interact with the silver surface. Therefore, SERS signals of benzene at silver substrates could be observed only at comparatively high benzene concentrations. For a bare metallic substrate immersed in a 10 mM benzene solution, the strongest benzene SERS band at 992 cm-I had a height of about 10% of the continuous background of the SERS substrate itself. The continuous inelastic background is generally observed together with SERS,I5and it limits the detection of weak adsorbate bands rather than their scattering intensity. For a BCAT-coated SERS substrate, the same ratio of the intensity of the 992 cm-I band and the background was obtained with 1mM solution. Unfortunately, a simple comparison of band intensities does not provide a proper measure of surface coverages with and without BCAT, because SERS enhancement strongly depends on the distance and chemical interaction between molecule and metal. Deiinitely, the BCAT-complexed benzene is positioned a certain distance from the metal, and thus, the enhancement should be weaker than that for molecules that are in direct contact with the bare metal surface. The SERS spectrum of a BCAT-coated substrate immersed in benzene solution (Figure 4a) is apparently a superposition of the SERS spectrum of the BCAT-coated substrate (Figure 4b) and (15) Akeman, W.; Otto, A. Surf: Sci. 1994, 307-309, 1071-1075
the Raman spectrum of benzene (Figure 4c). No indications for spectral changes due to the interactions between BCAT and benzene can be observed. Chlorobenzene and 1,2-dichlorobenzenehave also been com plexed with the BCATcoated substrates, giving SERS spectra with high signal to noise ratio even for substrates immersed into micromolar solutions. At bare metal substrates, 1,2dichlorobenzene was not observable even in nearly saturated solution, and the strongest SERS band of chlorobenzene could be distinguished from the background only in 2 mM solution. As with benzene, the SERS spectra of BCAT-coated substrates immersed in chlorobenzene (Figure 5) and 1,Zdichlorobenzenesolutions (Figure 6) are merely superpositions of the spectra of the substrate with that of the adsorbates without any indication of spectral changes due to complex formation. Alternate immersion of BCAT-coated substrates into solutions of the aromatics and pure water showed that the adsorption of all three investigated compounds is completely reversible. With solutions of higher concentrations,measurements with integration times of 10 s showed that the adsorption is practically complete after about 10 s. SERS Enhancement by Iodide. Iodide is known to give a substance-specific additional enhancement of SERS intensities.* For the BCATcoated substrates, it only slightly affected the BCAT intensities, but the intensities of aromatics adsorbed at BCATcoated substrates were increased 2-3-fold. Furthermore, the Analytical Chemisfry, Vol. 67,No. 78, September 75, 7995
3189
am-
1600
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1200
lo00
800
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400
Wavenrrmber Shift (om-1) Figure 4. SERS spectra of a BCAT-coated substrate within a 5 mM aqueous benzene solution (a) and within pure water (b). The Raman spectrum of benzene (c) is given for comparison. The SERS spectra are shifted upward.
120001
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f
am-
a
8
v
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60004000-
20000-
h
1600
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, aoo
L 600
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Wavenumber Shift (-4) Figure 5. SERS spectrum of a KAT-coated substrate within a 0.5 mM aqueous chlorobenzene solution (a). The Raman spectrum of chlorobenzene (b) is given for comparison. Spectrum a is shifted upward.
broadband background signal was reduced by the iodide. Altogether, the signal to noise ratio in the detection of aromatics was clearly improved. Therefore, iodide addition was included in the standard procedure for the measurements discussed further below. Concentration Dependence. The concentration dependencies of the analyte to BCAT peak height ratios are given in Figure 7. The detection limits derived were 100pM for benzene, 50 pM for chlorobenzene, and 5 pM for 1,Z-dichlorobenzene in water. Further lowering of these values should be straightforward by use of a holographic filter with high throughput instead of the two subtractively coupled monochromators for stray light reduction, by reduction of the dispersion of the spectrometer, and by evaluation of peak areas instead of heights. The calibration functions are roughly linear, as is expected for the lowconcentration part of Langmuir isotherms. Complexation of single analyte molecules within BCAT cages should strongly suppress interactions between the adsorbed analyte molecules. Therefore, the observed Langmuir isotherm should necessarily 3190 Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
appear for this type of adsorption. No saturation of the surfaces was observed up to solution concentrations near saturation. The dynamic range of the observable concentration dependencies was about 2 orders of magnitude. It was limited at higher concentrations by the solubility of the aromatic compounds in water and at lower concentrations by the broadband background signal from the substrate. The absolute intensities of the SERS bands scatter by some 10%for different substrate positions. Apparently this is caused by a somewhat inhomogeneous distribution of alumina particles on the glass slides and, therefore, a position dependence of the SERS enhancement. The use of the BCAT band intensities as an internal standard clearly reduces the dependence of aromatic intensities on the substrate properties and gives the reasonably linear calibration functions shown in Figure 7. This good linearity indicates that the adsorption at the coated substrate might include a stoichiometric complexation of the aromatics with the surfacebound BCAT.
wavenumber shift (om-1) Figure 6. SERS spectrum of a BCAT-coated substrate within a 0.25 mM aqueous 1,2-dichlorobenzenesolution (a). The Raman spectrum of 1,2-dichIorobenzene (b) is given for comparison. Spectrum a is shifted upward.
1
-2
'
,
-2
0 log (concentration / mM) -1
,
1
I
Figure 7. Concentration dependence of the SERS band height ratios of aromatic bands to the BCAT band at 1043 cm-I. For benzene ( x ) and chlorobenzene (0),the strongest bands, at 992 and 1002 cm-l, respectively,were used. For 1,2-dichIorobenzene(a),the band at 661 cm-' was chosen because of the overlap of the strongest band, at 1039 cm-l, with a BCAT band.
The slope of the concentration curves in the double logarithmic plot is somewhat smaller than the value of 1 expected for the surface coverage from Langmuir adsorption and slightly dependent on the adsorbed substance. A possible explanation for this effect may be that the plotted peak height ratios were not exactly proportional to the surface coverages with the analytes. For instance, the BCAT band height used as a reference may be somewhat enlarged by the adsorption of aromatics by an influence of the guest molecule on the structure, orientation, or polarizability of the host BCAT molecule. The lowering of the detection limits with increasing number of chloro substituents from 0 to 2 seems to point either to an interaction between BCAT and these substituents or to an inverse relationship between adsorption and solubility, as was observed for chlorinated ethylenes at octadecanethiol-coated substrates.6 However, a direct comparison of surface concentrations of the different compounds cannot be given, since the band intensities depend on the polarizabilities of the considered molecules, and the peak intensity ratios give, therefore, only a relative measure for the surface coverage. Attempts to detect hexachlorobenzene at modified substrates immersed in a saturated aqueous solution of this analyte failed because analyte bands were completely missing in the SERS
2.9
3.1
3.3 1/T (1/1OOO K)
3.5
3.1
Figure 8. Temperature dependence of the band height ratio of the chlorobenzene band at 1001 cm-' and the BCAT band at 1043 cm-I observed for a BCAT-coated substrate immersed in a 1 mM aqueous chlorobenzene solution.
spectra. This shows that the adsorption at the BCATcoated substrate is neither exclusively an inverse function of solubility, which is clearly lower for hexachlorobenzene than for the other investigated analytes, nor related only to an affinity between the chloro substituent and the BCAT cage. The missing hexachlorobenzene adsorption rather indicates that adsorption within the BCAT cage is a shape or size-selective process. Temperature Dependence. Absolute surface coverages are not directly measurable by SERS, since the Raman scattering intensities at the surface strongly depend on the unknown orientation of the adsorbed molecules. Therefore, the adsorption equilibrium constants for the analytes cannot be derived from our measurements. However, adsorption enthalpies can be obtained from changes of relative surface coverages due to temperature variation. An investigation of the dependence of the chlorobenzene to BCAT peak height ratio on the temperature was carried out in the temperature range between 2 and 70 "C. By always employing peak height ratios for quantification of the analytes, temperature influences on the SERS effect should be largely excluded, and the ratios should be good estimates for relative surface coverages with chlorobenzene. In the logarithmic plot of the peak height ratio versus 1/T (Figure 8), a linear relationshipwas obtained, as expected for Langmuir adsorption far from saturation. The slope of the curve gives an adsorption enthalpy of -6.7 kJ/mol for Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
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In contrast, the benzene band intensity is clearly diminished with increasing chlorobenzene concentration, which points to a replacement of benzene by its chloro derivative. However, the stronger decrease of the benzene band intensity at lower chlorobenzene concentrations in comparison to higher concentrations cannot be explained by the simple model of a merely competitive adsorption.
/ O
Y 0
0.5 1 1.5 chlorobenzeaecoacentratioa(mM)
I
2
Figure 9. Dependence of SERS band height ratios of the benzene band at 992 cm-’ (@) and the chlorobenzene band at 1002 cm-’ (A) to the BCAT band at 1043 cm-l on the chlorobenzene concentration. The benzene concentration is fixed to 1 mM. Error bars represent the standard deviation of repeated measurements on the same substrate with repeated filling of the cell.
chlorobenzene at immobilized BCAT. This low enthalpy indicates merely van der Waals interaction and may be due to the comparatively small size of the BCAT cone, not forming an optimum “recipient” for the aromatic guest molecules. The enthalpy is somewhat below the typical mean value of van der Waals interaction, which is -20 kJ/mol. It should be mentioned, however, that the measured value represents the difference between complexation and solvation enthalpies of the molecules. Adsorption from Mixtures. Since the SERS bands of the adsorbed compounds clearly depend on the substituents, the adsorbed aromatics can be identified and mixtures of the aromatics can be analyzed using BCAT-coated substrates. For quantitative analysis, however, it is necessary to know the mutual influence of coadsorbed species. Figure 9 shows the benzene and chlorobenzene band intensities as a function of the chlorobenzene concentration at a k e d benzene content. The relative peak height of the selected band of chlorobenzene, which is supposed to interact more strongly with BCAT, depends linearly on its concentration regardless of the presence of benzene. This corresponds to the expected behavior for low benzene coverage of the surface.
3192 Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
CONCLUSIONS Coating of rough silver films with BCAT has produced S E E substrates that form reversibly complexes with benzene and two of its chloro derivatives. The detection limits of SERS for these compounds have been lowered considerably by this complexation and by the additional enhancement effect caused by iodide. The reversibility of the surface complexation opens the possibility to determine the aromatic compounds considered via adsorption from aqueous solution with a dynamic concentration range of about 2 orders of magnitude. The comparatively small adsorption enthalpy resulting for chlorobenzene indicates the predominant effectiveness of van der Waals forces. Concerning possible applications for chemical sensors, sub stance-specjfic SERS sensing could exhibit selectivity and multisubstance capabilities superior to those of most other sensor principles, in which selective adsorption is used in connection with detection of less specific parameters such as refractive index, layer thickness, mass, light absorption, or fluorescence. ACKNOWLEDGMENT This work was financially supported by the Ministerium fir Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Bundesministerium fiir Forschung und Technologie, Germany, the National Science Foundation, and the Robert A Welch Foundation. Received for review April 3, 1995. Accepted June 20, 1995.B AC950335C Abstract published in Advance ACS Abstracts, August 1, 1995.
