CorresDondence Anal. Chem. 1995, 67,3353-3356
Molecular-Specific Chromatographic Detector Using Modified SERS Substrates Keith T. Cawon* and Brian J. Kennedy Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071-3838
Preliminary results for a gas chromatography detector based on surface enhanced Raman scattering (SERS) spectroscopy are described. The method described uses a SERS substrate that has been modified with a propanethiol coating that creates a hydrophobic interface to promote adsorption. The coating also prevents oxidation of the silver surface and stabilizes the SERS effect. The detectorwas tested with a separation of benzene, toluene, ethyibenzene, and o,m,p-xylenesusing a capillary column. The results demonstrate that a SERS detector is able to spectrally separate the chromatographically unresolved and detect a mixture component, benzene, at the 40 ng mass detection level. The temperature dependence of the GC-SERS cell was examined, and it was shown how reversibility of the detector is related to temperature. Separation science techniques,when coupled with a molecularspecific detector, are capable of providing detailed and often complete chemical analyses of complex mixtures. For most forms of chromatographythis has been realized with mass spectroscopy (MS) and Fourier transform infrared spectroscopy (FT-IR) systems. Mass spectroscopy is the most popular method of detection for chromatography due to its sensitivity and selectivity. FT-IR also provides detailed molecular information through vibrational spectroscopy and has good sensitivity. The disadvantages of these techniques are their expense and complexity. In particular, the need to perform MS techniques in a vacuum and the problems associated with solvent interference in FT-IR chromatographies lead to complicated instrument designs. We have shown that it is possible to qualitate and quantitate mixtures using surface-enhanced Raman scattering (SERS) spectroscopy with modified substrates.' The unique concept of our detection method is to fine tune the chemical potential of a SERS substrate by coating the silver or gold with a chemically bonded monolayer that matches the chemical properties of the analyte. We have demonstrated this capability with selective detection of metal P H , ~and organics in aqueous solutions and with (1) Mullen, IC I.; Wang, D.; Hurley, G. L.; Carron, IC Spectroscopy 1992, 7, 24-32. (2) Carron, IC; Mullen, IC; Lanouette, M.; Angersbach, H. Appl. Spectrosc. 1990,44, 63-69. (3) Crane, L. G.;Heyns, J. B. B.; Sears, L. M.; Wang, D.; Carron, IC Anal. Chem. 1995,67,485-490. 0003-2700/95/0367-3353$9.00/0 0 1995 American Chemical Society
organics in the vapor p h a ~ e . ~Once . ~ the analyte has been adsorbed by the surface coating we are able to detect it with high sensitivity and selectivity using SERS. The coatings are based on thiol chemistry and in many cases represent self-assembled monolayers. The thiols are known to stronglychemisorb to silver and gold surfaces to form monolayers that possess supramolecular proper tie^.^ These have been shown to drastically modify the surface energy of metals and with the addition of chemical functionalities can be tuned to provide a specific chemical property such as pH dependence, chelation with metals, or hydrophobicity. It became apparent from our work with the detection of chlorinated ethylenes6 and aromatics that we had developed a method of detection that mimics the interfacial processes that occur in chromatographic separations. In this paper, we will describe our first results from modified SERS substrates using gas chromatographic (GC) separation and SERS detection and identification. The mixture we have chosen for this example is benzene, toluene, ethylbenzene, and o,m&xylenes 0. BTEX is of interest since it is a common environmental contaminantand can be difficult to resolve with rapid GC.8 We will show thermodynamic relationships indicating that the results obtained with GC can be translated to other chromatographic techniques. EXPERIMENTALSECTION
The GC-SERS detection was made possible through recent developments in both SERS technology and fiber-optic coupling of Raman scattering. The SERS methodology for preparing substrates has been described elsewhere.'j The coating used in this study was 1-propanethiol (99.9%, Aldrich). The SERS s u b strate was prepared from 0.1 mm thick silver foil (9996, Aldrich) etched in 30% nitric acid. The coating was applied by soaking the foil in a 1 mM ethanolic solution for several hours. Before use the foils were washed with ethanol and air dried. Our basic Raman system has been described previously.6 The addition of fiber optics is new and will be described here. Figure 1 shows a schematic for the system. The Kr+ laser (Spectra (4) Mullen, IC: Wang, D.; Crane, L.; Carron, K. Anal. Chem. 1992,64, 930936. (5) Cari-on, IC;Pietersen, L., Lewis, M. Enuiron. Sci. Technol. 1992,26,19501954. (6) Mullen, IC;Carron, K. Anal. Chem. 1994,66,478-483. (7) Whitesides, G.;Laibnis, P.Langmuir 1990, 6,87-95. (8) Xiang, Y.;Morgan, S.; Watt, B. J. Chromatogr. Sci. 1995,33, 98-108.
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Benzene
PC Workstation
GC-SERS Cell GC-SERS Cell
'-0
I\'I
$K -
RLP RLP Kr+ Laser
I
Figure I. Schematicdiagram lor our GC-SERS fiber-optic coupled system. The components are described in the text.
Physics Model 202%) was operated at 647 nm and was coupled into the optical fiber (OF1) with a Detection Limit Technology @LT) laser-&fiber coupler (LFQ? The laser power was adjusted to provide 40 mW of power at the sample. The laser excitation and Raman collection were coupled with a DLT remote luminescence probe (RLP)? The RLP was designed for use at 647 nm with a high-throughput dichroic beam spliner that acts as a highly reflective long-pass filter. Further filtering is provided with an additional long-pass filter at the output of the RLP. The laser excitation and Raman collection are made with an epiillumination design using a 50 mm,fl2 lens. The Raman excitation is coupled into an optical fiber (Om)and is transferred to a speftroscopy input coupler (SIC) from DLT? The SIC fl# matches the fiber with the spearograph and provides 2.36 fiber image magnification that optimizes s p e d resolution and throughput This produces optimal SIN from the CCD by " k i n g the number of pixels binned in the vertical direction while optimizing the overall throughput, The Raman spectrum was dispersed with an HR320 and detected with a Photometrics CCD 9OOO system. The spectra were collected and stored with a PC workstation that also controls the interface with the gas chromatograph. The gas chromatography was performed with a SIU 8610 B equipped with a thermal conductivitydetector W D ) . The SERS detector was used both in tandem with the TCD and as a standalone detector when connected directly to the column. The column was a bigh-capacity, 1.0 pm thick methyl 50% phenyl silicone coated, 15 m, 530 pm i.d. capillary column (CC) from Quadrex Corp. The He carrier gas was set to flow at 8 mL/min and an isothermal column temperature of 75 "C chosen for this study. Total injection volumes were 3 pL In this report we will present data for BTM (Aldrich). Loading of the column was not observed for this injection volume. The GC-SERS detection cell was fabricated out of aluminum. For this preliminary study, a simple cell strategy was used. Our design used to collect the data in this report had an input and exit port for capillary connections. For the data in this report we used the exit port for the effluent as an optical port (see Figure 1, inset). The optical port was large enough for ns to use our epiillumination probe for excitation and collection of the SERS signal. The SERS substrate was placed in the effluent flow of the
em-' Figure 2. Raman spectra of the BTEX components in the spectral region used for the GC-SERS study. All spectra were obtained neat under the same conditions as the GC-SERS study.
capillary column as shown in F i 1. A temperature study was performed by cooling the cell prior to the chromatographic measurement or heating with heat tape. In all experiments the capillary line to the cell and extending from the column oven was heated to the oven temperature with heat tape. RESULTS AND DISCUSSION The SERS coating used in this report was 1-propanethiol. In previous reports, we have described a variety of coatingsfor SERS substrates. The role of the coating is twofold. It protects the silver from oxidation, and it creates a specific chemical environment at the interface to promote selective adsorption. Since silver or its oxides have very high snrface energies, and therefore little af6nity for hydrophobic molecules such as BTEX, a hydrophobic coating will lead to enhanced adsorption and therefore improved detection limits. Our choice of 1-propanethiol was empirically based on the decrease in SERS with increasing chain length and the increase in stability with increasing chain length. We have found that the interfacial enhancement drops off roughly as n-l.=, where interfacial enhancement is the Raman enhancement of scattering from molecules adsorbed to the surface of the coating and n is the distance from the surface in carbon chain length units.'Q Figure 2 shows the Raman spectra in the fingerprintregion of the individual BTEX components used in this study. It can be seen that each one has characteristic vibrations that can be used to idenfify the adsorbate. We have found that for material physisorbed to coated SERS substrates the SERS spectra are equivalent to normal Raman spectra of the pure material, with the exception of small intensity variations. For the monosubstituted benzenes, the variation is due to the mass of the substituent and the characteristic vibrations of the substituent" The disub stituted, isomeric xylenes have very different spectra due to the large variations in symmetry and ring substitution pattems." The peaks that are labeled in this figure are also shown in Figure 3C to illustrate the spectral resolution of the components coming off of the column. The SERS spectrum of 1-propanethiol, in the spectral region used in this study, contains two large Raman features that are C-C stretching vibrations at 1025 and 1084 cn-', a large Raman (10) Dickey. M.;Carron, K. to be submitted to AroL chcnr.
(9) Schoen. C. h w F -
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Schachtschneider,I.; Snyder, R S ~ c t m b i n rAcln . 1963,29, 117-128.
for chromatographic detection. Benzene and toluene are chromatographically resolved while ethylbenzene,pxylene, m-xylene, and o-xylene are convolved or poorly resolved. The raw GC-SEE chromatogram is shown in Figure 3B. The experimentswere repeated five times with equivalent results each time. The chromatogram is dominated by Raman features associated with the coating though one can see features that correspond to adsorbates. The effluent peaks can be seen more clearly if a spectrum of the coating is subtracted, as is shown in Figure 3C. This figure demonstrates that the equivolume (0.5 p L each) mixture of BTEX can be resolved and identitied with GC-SEE. The thermodynamic relationship that best describes the surface detection process is
1. Benzene 2. Toluene 3. Ethylbenzene 4. p-Xylene 5. m-Xylene 6. o-Xylene
-log P* = log k roo
see
900
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+ cpi" - S0)/2.3RT
llQ0
C"
J
8i
o
1dOl
9oe
1100
C"
Figure 3. (A) chromatogram of BTEX obtained under the same conditions as our GC-SERS data using a thermal conductivity detector. (B) GC-SERS chromatogram of BTEX. The total acquisition time is 300 s.(C) GC-SERS chromatogram of BTEX with the coating blank subtracted. Peaks labeled correspond to BTEX peaks labeled in Figure 2.
band due to a C-S stretch of the trans configuration at 702 cm-I, methyl rocking at 891 cm-l, and a methylene rock at 777 cm-1.11J2 These bands can be seen in Figure 3B. They remain virtually constant in intensity throughout the separation and can be used as an internal reference for the quantitation of material adsorbed to the surface. Small variations were observed when a high coverage of benzene and toluene was present on the surface. We believe the intensity variation may be due to changes in the local index of refraction at the interface due to adsorption of material. The chromatogram of BTEX is shown in Figure 3A. The column that we chose is not ideal for BTEX separations but illustrates the superior resolving power of a spectral technique (12) Bryant, M; Pemberton, J.J. Am. Chem. SOC.1991, 113, 3629-3637.
where P* is the vapor pressure of the effluent, k is the adsorption coefficient, 'pio is the chemical potential of the adsorbate at 0.5 monolayers, ipo is the chemical potential of the effluent in a saturated vapor, R is the gas constant, and T is the temperature in degrees kelvin. This can be derived in the same fashion as our linear free energy relationship for adsorption from solution.6 It contains two pertinent parameters, vapor pressure and adsorp tion coefficient. The adsorption coefficient is inversely related to vapor pressure and in chromatography retention time is directly related to k. In other words, the lower the vapor pressure the longer the retention time. This leads to the rule of thumb that lower boiling point materials (higher vapor pressures) will elute from a column first. The intercept in eq 1 takes into account differential adsorption of material due to surface chemical potentials created by different column coating polarities. It is the intercept that allows one to predict the column coatings required for separation of different materials. This common relationship for chromatography described above leads to an interesting result for a surface detection method like GC-SEE. The amount of material detected is ideally given by
e = k@/(i + kp)
(2)
where 0 is the fractional coverage and p is partial pressure of the adsorbate. In chromatography, since p decreases with retention time due to band broadening, volummetric detectors have lower detectabilities for strongly retained material. The GC-SERS detector is interfacial and obeys the relationship in eq 1. Therefore, if the coating is chemically equivalent to or similar to that of
10 "C
740 C"
Figure 4. GC-SERS detection of o-xylene.
I
"c
I
chromatogram of the 730-750 cm-' region of the spectrum. This figure illustrates the temperature dependence for the Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
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the column material, the detectability will stay constant throughout a separation. This is seen in Figure 3C, where the intensity of the o-xylene is not diminished relative to the benzene peak in comparison with the chromatogram obtained with a TCD detector shown in Figure 3A. This will make GC-SERS detection much more sensitive than current methods of detection for compounds that are highly retained. The GC-SERS chromatogram shown in Figure 3C was formed at 22 "C. Another potentially signifcant advantage of GC-SERS is the ability to temperature program the detector. Programming the detector temperature from low to high during a separation could be used to produce a high coverage of material without sacrificing reversibility. The effect of temperature on the detection of o-xylene is shown in Figure 4. It can seen that at 10 "C the o-xylene is retained on the detector, at 22 "C it is showing reversibility, and at 75 "C (the column temperature) the o-xylene is rapidly removed from the detector and the GC-SERS chromatographic bandwidth is determined by the band broadening within the column. A rough detection limit for benzene was determined from the 10 s integration period surrounding the benzene GC-SERS band and the point at which the signal to noise ratio is 3. We found a signal of 1185 analog-to-digital units (ADU) for the benzene peak and a noise level of 0.05 ADU, after smoothing, as determined by the standard deviation of 10 points along the background. Since our injection volume of benzene was approximately 0.5 pL, this gives a detection limit of about 50 ng. CONCLUSION
We have demonstrated that SERS can be coupled with a chromatographic separation to provide a sensitive and selective
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method of molecular detection. Since the adsorption mechanism is a thermodynamically driven process, the results from GC in this report will be generally applicable to other separation science methods. For example, in GC the relevant parameter is temperature; for HPLC this would be replaced by solvent polarity, and for supercritical fluid techniques this would be replaced by fluid density. Since the surface coverage determines the detection limit and this is related to the concentration (not total mass) through the isotherm eq 2, SERS methods should have equal sensitivity for microtechniques as well as preparation-scale separations. In particular, CE-SERS will become a very important separation science method given that capillary electrophoresis uses very low masses in the separation, but high concentration due to the small volumes used. SERS methods would also be particularly advantageous for separation methods that use aqueous mobile phases since these interfer strongly with FT-IR and MS detectors. ACKNOWLEDGMENT
The authors acknowledge the support of the NSF-EPSCoR ADP Grant EHR-9108774 for partial support of this project. We also acknowledge the support of Detection Limit Technology, LC Laramie, WY, for use of their RLP and SIC fiber-optic accessories. Finally, we acknowledge the support of the University of Wyoming for funding of patent applications. Received for review May 22, 1995. Accepted July 18, 1995.N AC9504880 @Abstractpublished in Advance ACS Abstracts, August 15, 1995.
