Anal. Chem. 2005, 77, 5965-5972
Reduction of Detection Limits of the Direct Deposition GC/FT-IR Interface by Surface-Enhanced Infrared Absorption David A. Heaps and Peter R. Griffiths*
Department of Chemistry, University of Idaho, Moscow, Idaho 83844-2343
Even though the interface of gas chromatography (GC) and mass spectrometry (MS) is by far the most popular way of identifying molecules eluting from a GC in real time, the identification of compositional isomers by MS is equivocal at best. Much better results would be found by infrared spectrometry (IR) if the sensitivity of the GC/IR interface could be improved. In this paper, we show how the smallest quantity of molecules injected into a GC for which an identifiable infrared spectrum can be measured on-line has been reduced by a factor of 10 below the detection limit of the most sensitive current technique. A commercial direct deposition interface between a GC and a Fourier transform infrared spectrometer was modified by vapor-depositing an island film of silver on the surface of the zinc selenide substrate. Band intensities in the spectra of molecules located within ∼4 nm of the surface of the silver islands were increased by at least 1 order of magnitude through surface-enhanced infrared absorption (SEIRA). The effectiveness of this approach was illustrated by comparing the limits of identification of butylbenzene isomers measured with and without the silver film. Comparison with the spectra of the same molecules measured by mass spectrometry showed the increased sensitivity and specificity of the GC/SEIRA interface. Any spectroscopic technique that is to be considered for the on-line identification of molecules eluting from a gas chromatography (GC) column must be fast, sensitive, and selective. Electron ionization (EI) mass spectrometry (MS) is certainly fast and sensitive and, for many compounds, gives a unique fragmentation pattern. By comparing measured mass spectra of GC peaks with the spectra in large spectral databases, many components of complex mixtures can be readily identified. The identification of GC peaks by mass spectral library searching has two drawbacks, however. EI mass spectra can vary with the energy of the ionizing electrons; for this reason, mass spectral libraries often contain multiple entries for a given compound. In addition, the mass spectra of constitutional isomers can be very similar, especially for aromatic molecules. For example, it is well known that the EI mass spectra of o-, m-, and p-xylene are essentially identical when measured under identical conditions.1,2 Even when fragmentation occurs on the substituent, as it does for the butylbenzene isomers * To whom correspondence should be addressed. E-mail: uidaho.edu. 10.1021/ac050585l CCC: $30.25 Published on Web 08/11/2005
pgriff@
© 2005 American Chemical Society
Figure 1. Reference mass spectra of the butylbenzene isomers for the on-line NIST database. (A) isobutylbenzene, (B) n-butylbenzene, (C) sec-butylbenzene, and (D) tert-butylbenzene.
that we discuss below, it may still be difficult to assign the correct structure to a given GC peak. It is well known that differences between the infrared spectra of constitutional isomers are usually much greater than the corresponding mass spectra. As an example, let us compare the mass spectra and infrared spectra of the four butylbenzene isomers. The mass spectra of these molecules contained in the NIST/EPA/NIH database3 are shown in Figure 1. It can be seen that the spectra of isobutylbenzene and n-butylbenzene are very similar and quite different from the spectra of sec-butylbenzene and tert-butylbenzene. The infrared spectra of these four isomers are shown in Figure 2. The substitution pattern may be determined from the strong bands absorbing between 900 and 650 cm-1. The strong bands at ∼750 and ∼700 cm-1, assigned to the aromatic C-H out-of-plane deformation mode and a ring-puckering vibration, respectively, immediately show that the aromatic ring is either monosubstituted (1) Wilkins, C. L.; Giss, G. N.; Brissey, G. M.; Steiner, S. Anal. Chem. 1981, 53, 113-117. (2) Crawford, R. W.; Hirschfeld, T.; Sanborn, R. H.; Wong, C. M. Anal. Chem. 1982, 54, 817-820. (3) NIST/EPA/NIH Mass Spectral Reference Database 1, National Institute of Standards and Technology, Gaithersburg, MD, 2002.
Analytical Chemistry, Vol. 77, No. 18, September 15, 2005 5965
Figure 2. DD GC/FT-IR of 30 ng of the butylbenzene isomers deposited on bare ZnSe at -95 °C measured in 1 s. (A) tertbutylbenzene, (B) n-butylbenzene, (C) sec-butylbenzene, and (D) isobutylbenzene.
or (with lower probability) 1,3-disubstituted.4,5 The spectra in the aliphatic C-H stretching region (3000-2800 cm-1) readily distinguish between the number of CH2 and CH3 groups in the butyl group. Thus tert-butylbenzene, with three CH3 groups and no CH2 groups is readily distinguished from n-butylbenzene, with its one CH3 group and three CH2 groups. However, the spectra of isobutylbenzene and sec-butylbenzene are very similar in this region, because each has two CH3 groups and one CH2 group. These two compounds may be distinguished by the fact that the two CH3 groups in isobutylbenzene are attached to the same carbon atom so that some of the vibrations of these CH3 groups are coupled, whereas this is not the case for sec-butylbenzene. As a result, the symmetric CH3 deformation band at ∼1380 cm-1 is split, while this mode is a singlet in the spectrum of secbutylbenzene. The spectra of the four butylbenzene isomers in this region are shown in Figure 3. In the first, and still the most commonly used, approach to GC/FT-IR, the sample is passed in the vapor phase through a heated light-pipe gas cell.6-8 To identify each component, the measured spectrum is compared to a library of vapor-phase spectra, for which the largest database (Aldrich) contains fewer than 10 000 reference spectra. Detection limits in infrared spectrometry for different molecules can vary by at least 1 order of magnitude because the absorptivity of bands in IR spectra depends on the change in dipole moment during the vibration. Thus, the limit of detection (LOD) of nonpolar molecules (such as the butylbenzene isomers on which we report in this paper) may be (4) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: San Diego, CA, 1991. (5) Mayo, D. W.; Miler, F. A.; Hannah, R. W. Course Notes on the Interpretation of Infrared and Raman Spectra; Wiley-Interscience: New York, 2004. (6) Azarraga, L. V. Appl. Spectrosc. 1980 34, 224-226. (7) Herres, W. HRGC-FTIR: Capillary Gas Chromatography Fourier Transform Infrared Spectroscopy, Theory and Applications; Huethig: Heidelberg, Germany, 1987. (8) Reedy, G. T.; Bourne, S.; Cunningham, P. T. Anal. Chem. 1979, 51, 15351540.
5966 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005
Figure 3. ATR spectra of the butylbenzene isomers in the spectral range between 1420 and 1320 cm-1. (A) tert-butylbenzene, (B) n-butylbenzene, (C) sec-butylbenzene, and (D) isobutylbenzene.
much greater than the LOD of polar molecules. With a light-pipe GC/FT-IR interface, the LOD is typically between ∼50 (for nonpolar compounds) and ∼10 ng (for polar compounds.) The sensitivity of GC/FT-IR measurements was increased beyond that obtained with a light-pipe by isolating each separated component in a matrix of solid argon in the manner developed by Reedy et al.8-10 Real-time measurements were not possible with the matrix isolation (MI) GC/FT-IR interface, and the spectrum of each analyte had to be measured after all components had eluted. Because the samples are trapped in an area that is smaller than the cross-sectional area of a light-pipe, detection limits with the MI GC/FT-IR interface are at least a factor of 10 lower than for the light-pipe interface. Again, however, a special-purpose library, this time of MI spectra, was required; this database contained significantly fewer entries than the library of vapor-phase spectra. This factor, in combination with the mechanical complexity of the MI GC/FT-IR interface, led researchers to consider other approaches. The third, and most sensitive, type of GC/FT-IR interface relies on the direct deposition of the effluent from the GC column onto a slowly moving, liquid nitrogen (LN2)-cooled zinc selenide window so that each component condenses as a very small spot on the window.10-13 Immediately after the deposition, each spot passes through the beam of a simple FT-IR microscope operating in the transmission mode, so that spectra are measured at intervals of 1 s or less. The smaller the diameter of the spot, the thicker is the sample and the greater the peak absorbance. Because the diameter of each deposited component can be made as small as ∼100 µm, the amount of polar molecules required to give a spectrum in real time (i.e., as the spot passes through the infrared (9) Reedy, G. T.; Ettinger, D. G.; Schneider, J. F.; Bourne, S.; Cunningham, Anal. Chem. 1985, 57, 1602-1609. (10) Visser. T. Gas Chromatography/Fourier Transform Infrared Spectroscopy. In Handbook of Vibrational Spectroscopy; Chalmers, J. M., Griffiths, P. R., Eds.; John Wiley and Sons: Chichester, U.K., 2002; Vol. 2, pp 1605-1626. (11) Haefner, A. M.; Norton, K. L.; Griffiths, P. R.; Bourne, S.; Curbelo, R, Anal. Chem. 1988, 60, 2441-2444. (12) Bourne, S.; Haefner, A. M.; Norton, K. L.; Griffiths, P. R. Anal. Chem. 1990, 62, 2448-2452. (13) Norton, K. L.; Griffiths, P. R. J. Chromatogr., A 1995, 703, 383-392.
Figure 4. Infrared spectra of (A) 30 ng of isobutylbenzene on ZnSe at -95 °C measured on-line in 1 s; (B) liquid film of isobutylbenzene between two NaCl plates at 20 °C.
beam) is usually less than 500 pg. Jackson et al.14 and Visser10 have compared the GC/MI-FT-IR and GC/direct-deposition (DD)FT-IR interfaces and showed that, for spectra measured in equal times, the sensitivity of the DD interface is between a factor of 5 and 10 greater than that of the MI interface. It should be recognized, however, that only the GC/DD-FT-IR interface is capable of measuring the spectra of separated components in real time. GC/FT-IR spectra measured by the DD technique have an additional advantage in that the spectra are often very similar to the standard reference spectra of solid samples prepared as KBr disks12,13 or even liquid samples run as capillary films. As an example, the DD GC/FT-IR spectrum of isobutylbenzene at -95 °C is compared to the liquid-phase spectrum of the same molecule at ambient temperature in Figure 4. Because, for DD GC/FT-IR, standard reference spectra of condensed-phase samples may be used, the number of compounds for which reference spectra are available is greatly increased, since libraries of condensed-phase spectra exceed 100 000 entries. With all early GC/FT-IR interfaces, the amount of sample that had to be injected onto a GC column in order for a given component to be identified, which we will call the limit of identification (LOI), was usually at least 100 times greater for GC/ FT-IR than for GC/MS. This large difference in sensitivity has led most GC practitioners to rely on MS as the sole technique used for the on-line identification of all peaks in their chromatograms. Indeed, many chromatographers have complete faith in the results of a GC/MS library search to the point and assign the identity of each peak to the first “hit” in the list of possible matches without performing a visual comparison of the measured spectrum and the best match. A reliable GC/IR interface with the same detection limits as even a benchtop GC/MS interface would greatly increase the reliability to which each component of a mixture that has been separated by gas chromatography could be identified. The goal of this project is to decrease the detection limits of DD GC/FT-IR measurements by taking advantage of the phe(14) Jackson, P.; Dent, G.; Carter, D.; Schofield, D. J.; Chalmers, J. M.; Visser, T.; Vredenbregt, M. J. High Resolut. Chromatogr. 1993, 16, 515-521.
nomenon of surface-enhanced infrared absorption (SEIRA)15,16 in an analogous way to the off-line measurements reported for liquid chromatography by Sudo et al.17 Our goal was to reduce the LOI for spectra measured on-line using the DD GC/SEIRA interface to the point that it is equal or less than that of GC/MS. Even though SEIRA has been recognized for 25 years,18 it has found far fewer applications to date than its vibrational spectroscopic counterpart, surface-enhanced Raman scattering (SERS). In SEIRA, the absorbance of molecules within a few nanometers of the surface of island films of silver and gold is enhanced by up to ∼2 orders of magnitude, which is ∼100 times less than for SERS in the absence of resonance enhancement. Other metals also give rise to surface enhancement of infrared spectra, but usually by only a factor of ∼10.19-23 In this paper, we report how coating the surface of the ZnSe substrate with a thin island film of silver can reduce the detection limit of the GC/FT-IR interface to the point that it becomes comparable to those of GC/MS. SEIRA spectra must be essentially identical to standard DD GC/FT-IR spectra if molecules are to be identified by spectral searching. Several research groups have reported22-26 that the shape of bands in the SEIRA spectra of species on the surface of metal-island films can become asymmetric under certain circumstances. Avoiding this asymmetric band shape is important if a DD GC/SEIRA spectrum is to be input to a standard database for spectral library searching. EXPERIMENTAL SECTION A Bourne Scientific (Newton, MA) infrared chromatograph (IRC) DD GC/FT-IR interface was used to obtain all GC/FT-IR spectra shown in this paper.27 In this instrument, the effluent from the GC is passed through a heated transfer line through a fusedsilica deposition tip onto a cooled ZnSe window the position of which is controlled by an x-y stage. The beam of a rudimentary FT-IR microscope (beam diameter, 100 µm) is located less than 1 mm from the deposition tip so that the spectrum of each component is measured very shortly after it is deposited. To avoid the possibility of sample condensation, the temperature of the transfer line was held 10 °C above final temperature of the GC program. The temperature of the deposition tip was set at 250 °C. The temperature of the ZnSe substrate was thermostated at -95 °C, with the pressure in the vacuum chamber at ∼5 × 10-5 Torr. Thin films of silver were deposited using a custom-built physical vapor deposition apparatus. For the deposition of silver, (15) Osawa, M. Bull. Chem. Soc. Jpn. 1997, 70, 2861-2880. (16) Aroca, R. F.; Ross, D. J.; Domingo, C. Appl. Spectrosc. 2004, 58, 324A338A. (17) Hartstein, A.; Kirtley, J. R.; Tsang, J. C. Phys. Rev. Lett. 1980, 45, 201-4. (18) Sudo, E.; Esaki; Y.; Sugiura, M. Bunseki Kagaku 2001, 50, 703-707. (19) Bjerke, A. E.; Griffiths, P. R. Appl. Spectrosc. 2002, 56, 1275-1280. (20) Bjerke, A. E.; Griffiths, P. R; Theiss, W. Anal. Chem. 1999, 71, 19671974. (21) Chen, Y.-J.; Sun, S.-G.; Chen, S.-P.; Li, J.-T.; Gong, H Langmuir 2004, 20, 9920-9925. (22) Wang, H.-C.; Sun, S.-G.; Yan, J.-W.; Yang, H.-Z.; Zhou, Z.-Y. J. Phys. Chem. B 2005, 109, 4309-4316. (23) Krauth, O.; Fahsold, G.; Magg, N.; Puuci, A. J. Chem. Phys. 2000, 113, 6330-6333. (24) Ishida, K. P.; Griffiths, P. R. Anal. Chem. 1994, 66, 522-530. (25) Merklin; G. T.; Griffiths, P. R. J. Phys. Chem. B 1997, 101, 5810-5813. (26) Brown, C. W. University of Rhode Island. Personal communication to P.R.G., 2003. (27) Bourne, S. Am. Lab. 1998, 30 (16), 17F-17J.
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Table 1. Results of Searching GC/MS Spectra of the Butylbenzene Isomers Injected at Levels of 2.9 ng compound
hit rank
molecule in MS “hit list”
direct match factor
reverse search factor
probability factor
isobutylbenzene
1 2 3 4 5 6 7 8 9 10
isobutylbenzene n-butylbenzene isobutylbenzene acetophenone, 4-(benzyloxymethyl)isobutylbenzene n-butylbenzene bicyclo[2.2.2]oct-7-en-2-one, 5-methyleneisobutylbenzene n-butylbenzene n-butylbenzene
598 582 573 570 568 568 561 561 560 559
641R 622R 616R 607R 611R 610R 595R 622R 590R 599R
26.3P 15.2P 26.3P 10.1P 26.3P 15.2P 7.33P 26.3P 15.2P 15.2P
n-butylbenzene
1 2 3 4 5 6 7 8 9 10
n-butylbenzene n-Butylbenzene n-butylbenzene n-butylbenzene n-butylbenzene isobutylbenzene isobutylbenzene isobutylbenzene 1,2,3,4,4a,8a- hexahydronaphthalene 1-hexen-4-ol, 3-methyl-6-phenyl-
720 717 715 709 708 706 699 689 681 675
741R 743R 736R 744R 734R 732R 724R 714R 704R 680R
38.4P 38.4P 38.4P 38.4P 38.4P 24.1P 24.1P 24.1P 7.34P 5.77P
sec-butylbenzene
1 2 3 4 5 6 7 8 9 10
benzene, diethylbenzene, 1-methyl-3-propylsec-butylbenzene benzeneethanol, 3-methyln-butylbenzene hydrazine, (2-phenylethyl)benzene, 1-methyl-2-propylbenzene, 1-methyl-3-propyloxirane, 2-methyl-3-phenylbenzeneethanol, β-methyl-, (S)-
638 612 605 598 582 580 574 571 570 569
794R 719R 708R 752R 747R 585R 678R 608R 694R 574R
41.7P 12.5P 9.57P 7.33P 4.22P 3.89P 3.06P 12.5P 2.58P 2.48P
tert-butylbenzene
1 2 3 4 5 6 7 8 9 10
benzene, 1-methyl-4-(1-methylethyl)benzene, 1,2,4,5-tetramethylbenzene, 1,2,3,5-tetramethyltert-butylbenzene benzene, 1-methyl-4-(1-methylethyl)benzene, 1-methyl-2-(1-methylethyl)tert-butylbenzene tert-butylbenzene benzene, 1-methyl-3-(1-methylethyl)oxazole, 2,5-dihydro-5-(4-methylphenyl)-4-phenyl-
619 619 618 616 615 611 609 608 606 603
728R 709R 752R 731R 724R 716R 717R 736R 701R 716R
10.2P 10.2P 9.75P 9.00P 10.2P 7.25P 9.00P 9.00P 5.84P 5.16P
the vacuum chamber was first evacuated to 0.1 Torr and a plasma glow discharge was used to clean the ZnSe surface prior to the plating of the silver. The glow discharge was formed at 3 mA and 3 kV for 5 min at a pressure of 0.1-0.05 Torr. After the glow discharge was terminated, the pressure was reduced to 1 × 10-6 Torr with an oil diffusion pump. The amount of silver deposited was measured with a quartz crystal microbalance; the thickness and deposition rates given in this paper are calculated by assuming that the deposited layer is uniform. All results shown in this paper were obtained with a 5-nm-thick silver-island film deposited at a rate of 1.2 × 10-2 nm s-1. Separations were performed on a Hewlett-Packard 5890 II GC equipped with a 30:1 inlet splitter. The column was 15 m long with a 0.25-mm internal diameter (i.d.); the stationary phase was a 0.25-µm film of 5% methyl, 95% phenyl polysiloxane (ZB-5, Phenomenex). Most separations were performed by holding the column at 150 °C for 10 min, followed by a temperature ramp of 5 °C/min to a final temperature of 225 °C. The column head pressure was 10 psi. Solutions of the butylbenzene isomers (99% pure, Aldrich) in methanol at concentrations of 1500, 86, and 8.6 ppm solutions were prepared. For all separations reported in this paper, 1 µL of these solutions was injected. Since the GC was 5968
Analytical Chemistry, Vol. 77, No. 18, September 15, 2005
equipped with a 30:1 inlet splitter, 50 ng, 2.9 ng, and 290 pg of the butylbenzenes were deposited on the bare or silver-coated ZnSe window. GC/MS analyses were performed by injecting 1.0-µl aliquots of each solution into a Hewlett-Packard Model 6890 chromatograph equipped with a 30:1 inlet splitter and a 30-m, 0.25-mm-i.d. column with a 0.25-µm film of DB-5. The column was held at a temperature of 50 °C for 2 min and then increased at the rate of 15 °C/minute until all components had eluted. The output of the column was passed into a JMS-AX505HA mass spectrometer (JEOL) that was scanned from 30 to 300 m/z every 1.0 s, with unit resolution. The mass spectrometer was operated with an ionization voltage of 70 eV, source temperature of 180 °C, and detector voltage of 2 kV. RESULTS Mass spectra are usually identified by a combination of a forward search and a reverse search.28 In the forward search, a similarity value is calculated that is a measure of how closely the intensities of a certain number (often 5 or 10) of lines in the sample (28) Stein, S. E.; Scott, D. R. J. Am. Soc. Mass Spectrom. 1994, 5, 859-866.
spectrum match the intensities of the corresponding lines for all library spectra. In reverse searches, the intensities of the lines in the library spectra are compared to the intensity of the corresponding lines in the sample spectra. Forward searching generally works best when the GC peak contains only one component, whereas a reverse search is more useful if a component with a similar retention time coelutes with the GC peak of interest. Some library searching programs include an algorithm that gives a probability factor that a given peak has been correctly identified. If the library contains several entries for a given molecule and more than one of these entries are found to be in the top 10 matches, the probability of a correct identification is increased. If, on the other hand, only one reference spectrum of a given molecule is contained in the library, the probability factor of a correct match is reduced. To illustrate some of the limitations of contemporary mass spectral library searching algorithms, GC/MS spectra of 2.9 ng each of the four butylbenzenes were measured. There are five reference spectra each of n-butylbenzene and sec-butylbenzene and four spectra each of isobutylbenzene and tert-butylbenzene in the MS database. The results of library searching are shown in Table 1. It can be seen that the first five hits for n-butylbenzene were each of the authentic compound, despite the great similarity of the spectra of n-butylbenzene and isobutylbenzene seen in Figure 1. Not unexpectedly, the next three hits were also for isobutylbenzene. In light of the similarity of the reference spectra of n-butylbenzene and isobutylbenzene, this result could certainly be classified as a success. The results for isobutylbenzene were more equivocal, with isobutylbenzene appearing as hit ranks 1, 3, 5, and 8 and n-butylbenzene as hit ranks 2, 6, 9 and 10. Despite the spectral similarity to n-butylbenzene, isobutylbenzene was successfully identified. We expected the results for sec-butylbenzene and tert-butylbenzene to be even better, since the mass spectra of these compounds are quite different from each other and from the spectra of the n and iso isomers (see Figure 1). However, this was not found to be the case. sec-Butylbenzene was identified as 1-methyl,3-propylbenzene, with diethylbenzene as the second match and sec-butylbenzene third. Note that even though all three compounds are isomeric with monosubstituted butylbenzenes, the first two hits are disubstituted molecules; the probability factor that the compound was in fact sec-butylbenzene was less than 10%. Even worse results were found with tert-butylbenzene, which was ranked as having the fourth highest probability, with the more highly ranked compounds being either di- or tetrasubstituted. Only the fact that there are four reference spectra of tert-butylbenzene in the database caused the probability of this compound being identified correctly to be as high as it was. Despite the uncertain identification of the butylbenzene isomers by GC/MS and the expectation that infrared spectrometry would allow these isomers to be distinguished with much more certainty than MS, the low sensitivity of IR spectrometry leads the vast majority of gas chromatographers to use MS as their primary peak identification tool. As noted above, infrared spectra allow many constitutional isomers to be identified unequivocally but the detection limits of GC/FT-IR interfaces are usually far worse than those on GC/ MS, especially when the analytes are nonpolar. For example, each
Table 2. Results of Searching DD GC/FT-IR Spectra of the Butylbenzene Isomers Injected at Levels of 2.9 ng compound n-butylbenzene
sec-butylbenzene
hit rank 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
tert-butylbenzene
1 2 3 4 5 6 7 8 9 10
molecule in IR “hit list”
match
n-butylbenzene amylbenezene propylbenzene tetraphenylethylene benzhydrol stilbene oxide, transtriphenylmethane m-terphenyl triphenylphosphine benzyl sulfide
89.28 86.84 81.47 77.33 76.85 76.70 75.73 73.34 71.31 70.75
sec-butylbenzene cumene poly(styrene-acrylonitrile), 25% acrylonitrile 3-phenyl-1-butanol poly(styrene-vinylidene chloride) tert-butylbenzene benzyl mercaptan ABS plastic (ATR Ge crystal) ABS plastic (ATR corrected) (+)-2-phenyl-1-propanol
78.18 77.51 74.85
tert-butylbenzene cumene sec-butylbenzene 3-phenyl-1-butanol poly(styrene-vinylidene chloride) poly(styrene-acrylonitrile), 25% acrylonitrile (+)-2-phenyl-1-propanol (()-1-phenyl-1-propanol benzyl chloride ABS plastic (ATR Ge crystal)
72.01 70.84 68.22 68.09 67.01 66.91 66.28 82.51 75.26 68.25 63.46 62.83 60.63 59.27 56.33 56.25 55.93
of the DD GC/FT-IR spectra shown in Figure 2 was of 29 ng of each butylbenzene isomer injected on the column. On-line DD GC/FT-IR spectra of equivalent quality of sample quantities as low as 300 pg of 3-ethylphenol12 and 75 pg of barbiturates13 have been reported previously. However, these molecules are far more polar than the butylbenzene isomers examined in this investigation, and consequently, the absorptivities of the stronger bands are significantly higher. Furthermore, the spectra in refs 12 and 13 were measured using the Bio-Rad Tracer, for which the baseline noise is a factor of almost 10 lower than noise measured in an equivalent time with the Bourne Scientific IRC. The result of searching these spectra against several libraries of condensed-phase infrared spectra (Aldrich Condensed Phase Sample Library, Georgia State Crime Lab Sample Library, Hummel Polymer Sample Library, Nicolet Condensed Phase Academic Sampler, and Sigma Biological Sample Library) is shown in Table 2. The reference spectrum of isobutylbenzene was not in these databases and so the results are not shown for this molecule. The corresponding result obtained when the concentration of the analytes was reduced by 1 order of magnitude is shown in Table 3. To increase the match factor for Table 3 (290-pg sample), spectra measured over a 5-s interval were averaged together. Even for the data shown in Table 3, for which the signal-to-noise ratio (SNR) of the input spectra was relatively poor, the authentic compound was the first hit, demonstrating the success of infrared spectrometry in identifying the correct isomer. Not surprisingly Analytical Chemistry, Vol. 77, No. 18, September 15, 2005
5969
Table 3. Results of Searching DD GC/FT-IR Spectra of the Butylbenzene Isomers Injected at Levels of 290 pg compound n-butylbenzene
sec-butylbenzene
hit rank 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
tert-butylbenzene
1 2 3 4 5 6 7 8 9 10
molecule in IR “hit list”
match
n-butylbenzene propylbenzene amylbenzene ethylbenzene diphenylmercury biphenyl triphenylphosphine triphenylarsine sec-butylbenzene benzphetamine in KBr
43.37 41.43 40.22 38.87 36.34 35.73 34.69 34.63 34.33 34.13
4-phenyltoluene 1-phenyl-1-propyne cumene sec-butylbenzene poly(styrene) poly(styrene) atactic tert-butylbenzene poly(styrene-acrylonitrile), 25% acrylonitrile benzyl mercaptan poly(styrene-vinylidene chloride)
39.13 36.79 36.77 35.77 31.81 31.34 31.21 28.95
benzonitrile benzonitrile phenylacetylene 1-phenyl-1-propyne tert-butylbenzene ethyl benzoate ethyl benzoate cumene p-chlorobiphenyl (E)-stilbene
29.95 29.95 26.42 25.97 25.40 23.61 23.60 23.51 23.05 22.84
Figure 5. Spectra of a self-assembled monolayer of p-nitrothiophenol adsorbed on silver films deposited on a ZnSe substrate at the same rate: (A) 5-nm-thick film; (B) 6-nm-thick film.
28.15 28.04
in view of the very strong characteristic bands at ∼750 and 700 cm-1, every single molecule listed in Tables 2 and 3 contains a monosubstituted aromatic ring. For n-butylbenzene, the second and third “hits” were n-amyl and n-propylbenzene, which illustrates that infrared spectroscopy has more difficulty in distinguishing between members of a homologous series than between individual substitutional isomers. It may also be noted that no n-alkylbenzene was ever listed among the top hits for the branched isomers. Last, and not surprisingly, by comparing the results shown in Tables 2 and 3, it is very clear that the match factor for the first hit, and hence the confidence that can be placed in the result, was far worse when the SNR was poor, varying from between 89 and 78 for the high-concentration sample to between 43 and 30 for the low-concentration sample. Even though the first compound listed in Table 3 was correct, a match factor value of less than 50 usually indicates either that the authentic reference spectrum is not in the library or that the SNR was too low to have any confidence in the result. It is also noteworthy that the reference spectra of the butylbenzene isomers were measured with the sample in the liquid phase at ambient temperature, while for the DD GC/FT-IR spectra, the samples were solid at -95 °C. The results shown in Tables 2 and 3 suggest that even higher match factors could be achieved if the SNR of the spectra could be increased above the level of the spectra shown in Figure 2. Since each separated component is condensed on the LN2-cooled substrate, one way to decrease the noise level of any spectrum is by postrun signal averaging. However, this approach would not 5970 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005
Figure 6. DD GC/FT-IR spectra of 2.9 ng of tert-butylbenzene on (A) 5-nm silver on ZnSe and (B) bare ZnSe.
yield real-time measurements. In this paper, we report the feasibility of increasing the SNR of on-line DD GC/FT-IR spectra by coating the ZnSe substrate with a thin island film of silver so that the absorbance of the analyte molecules that are located within ∼5 nm of the surface is significantly enhanced by SEIRA. Both the thickness and the morphology of the silver film must be optimized if the highest SNR is to be attained for SEIRA spectra of thin films of organic molecules. We have found that the thickness of the metal film and the rate at which it is deposited have an important effect on both the factor by which the band intensities of thin films of organic molecules are enhanced and the shape of the absorption bands.29 In general, as the nominal thickness of the metal film is increased from ∼1 to 10 nm, the enhancement factor increases by more than 1 order of magnitude. However, when the density of the metal islands reaches the point in which they start to coalesce, or percolate, the absorption bands of any molecule near the surface of the islands become asymmetrical, severely reducing the success of spectral library searching. The rate at which the silver is vapor deposited also has an effect on the thickness sat which percolation starts. At a deposition (29) Heaps, D. A.; Griffiths, P. R., unpublished results, 2003.
Table 4. Results of Searching DD GC/SEIRA Spectra of the Butylbenzene Isomers Injected at Levels of 2.9 ng compound n-butylbenzene
sec-butylbenzene
hit rank 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
tert-butylbenzene
1 2 3 4 5 6 7 8 9 10
molecule in IR “hit list”
match
n-butylbenzene amylbenzene propylbenzene benzhydrol tetraphenylethylene stilbene oxide, transm-terphenyl triphenylmethane triphenylphosphine sec-butylbenzene
83.62 80.59 78.09 68.10 68.09 67.97 67.03 65.99 65.35 63.80
sec-butylbenzene cumene 3-phenyl-1-butanol tert-butylbenzene ABS plastic (ATR Ge crystal) ABS plastic (ATR corrected) (()-1-phenyl-1-propanol (+)-2-phenyl-1-propanol poly(styrene-vinylidene chloride) poly(styrene-acrylonitrile), 25% acrylonitrile
83.22 78.78 70.73 69.83 65.94 63.16 61.87 61.48 60.88
tert-butylbenzene cumene sec-butylbenzene 3-phenyl-1-butanol (+)-2-phenyl-1-propanol tert-butylbenzene poly(styrene- vinylidene chloride) (()-1-phenyl-1-propanol ABS plastic (ATR Ge crystal) 2,2,4-trimethylpentane
81.67 71.86 63.49 59.40 54.26 50.03 49.36
59.97
Table 5. Results of Searching DD GC/SEIRA Spectra of the Butylbenzene Isomers Injected at Levels of 290 pg hit rank
molecule in IR “hit list”
match
n-butylbenzene
1 2 3 4 5 6 7 8 9 10
n-butylbenzene amylbenzene tetraphenylethylene octane stilbene oxide, transbenzhydrol triphenylmethane amylamine tetrabutylammonium bromide propylbenzene
76.41 75.82 70.43 69.46 68.55 67.84 67.58 67.10 66.20 65.69
sec-butylbenzene
1 2 3 4 5 6 7 8 9 10
sec-butylbenzene cumene tert-butylbenzene 3-phenyl-1-butanol tert-butylbenzene (()-1-phenyl-1-propanol 2,2,4-trimethylpentane o-diethylbenzene (1R,2S,5R)-(-)-menthol ethylbenzene
76.47 71.67 63.25 55.56 54.67 53.43 53.30 52.96 52.72 52.72
tert-butylbenzene
1 2 3 4 5 6 7 8 9 10
tert-butylbenzene cumene sec-butylbenzene tert-butylbenzene 2,2,4-trimethylpentane 3-phenyl-1-butanol cyclopentane 3,3-dimethylpentane 2,4,4-trimethyl-2-pentene o-diethylbenzene
73.72 67.90 61.52 59.03 54.55 48.56 48.28 46.66 44.56 43.95
compound
48.85 48.65 47.83
rate of ∼0.01 nm s-1, percolation of the silver islands starts when the thickness reaches ∼6 nm. As the deposition rate is increased, the onset of percolation starts at lower film thicknesses. For the measurements described here, silver was deposited at 1.2 × 10-2 nm s-1. At this deposition rate, a thickness of 5 nm of silver gave the maximum enhancement without percolation occurring. The spectra of a self-assembled monolayer (SAM) of p-nitrothiophenol (PNTP) prepared in the way described by Carron and Kennedy30 on 5- and 6-nm-thick silver films are shown in Figure 5. It can be seen that bands in the spectra of PNTP adsorbed on a 6-nm silver film are asymmetric while those on the 5-nm silver film are not, indicating the onset of percolation at a nominal thickness of 6 nm. A 5-nm island film of silver deposited on the ZnSe substrate of a DD GC/FT-IR interface reduced the LOI to below the level achievable with an uncoated ZnSe window. The DD-GC/SEIRA spectra of 2.9 ng of tert-butylbenzene measured with and without the silver film are shown in Figure 6. Note that the absorbance of corresponding bands in the spectra measured with the silver film has increased significantly because of surface enhancement. With 5-nm films, the baseline noise was increased by less than a factor of 2 because the transmittance of the silver film is greater than 50%. The results of searching the spectra of the three butylbenzene isomers whose spectra were available in the database that we used are shown in Table 4. (30) Carron, K. T.; Kennedy, B. J. Anal. Chem. 1995, 67, 3353-3356.
Figure 7. DD GC/FT-IR spectra of 290 pg of tert-butylbenzene on (A) 5-nm silver on ZnSe and (B) bare ZnSe.
Osawa showed that the absorption of only those molecules that are within 4 nm of the surface is enhanced.15 For 2.9-ng injections of n-, sec-, and tert-butylbenzene, the thickness of the sample is much greater than 4 nm, so that the effect of surface enhancement was minimal. The average value of the match factors obtained with and without the silver film was ∼83 in both cases. Thus, it is likely that for the thicker deposits, most of the spectral intensity originates from molecules that are more than 4 nm from the metal surface. Analytical Chemistry, Vol. 77, No. 18, September 15, 2005
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For the 290-pg injections, the average value of the match factors obtained on bare ZnSe was ∼37, while the corresponding values when the ZnSe substrate had been prepared with a 5-nm silver film was ∼76 (Figure 7). The significantly improved match factor of the latter spectra can be largely attributed to the increased SNR caused by SEIRA, but these high match factors could also only be achieved if the amount of spectral distortion introduced by the 5-nm silver film was negligible (Table 5). We also note that both the DD GC/FT-IR and GC/SEIRA spectra are very similar to the liquid-phase spectra in the database, allowing for library searching against existing condensed-phase libraries even when the sensitivity has been enhanced through the SEIRA effect. The intensity of bands in the reflection-absorption spectra of very thin layers of molecules on any metal surface is governed by the surface selection rule; i.e., only those vibrational modes that have a component of the dipole moment derivative that is perpendicular to the surface of the metal are infrared active. The same rule applies to SEIRA spectra, as can be seen by comparing the SEIRA spectrum of a SAM of PNTP to a spectrum of a KBr disk of the same molecule. In the spectrum of PNTP prepared as a KBr disk, the intensities of the symmetric NO2 stretching mode at 1340 cm-1 and the antisymmetric NO2 stretching mode at 1515 cm-1 are approximately equal. When PNTP is adsorbed onto a flat metal surface, the dipole moment derivative of the symmetric NO2 stretch is perpendicular to the surface of the silver islands, while for the antisymmetric NO2 stretch, the dipole moment derivative is approximately parallel to the silver surface. The
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intensity of symmetric stretching band is, therefore, considerably more intense than that of the antisymmetric stretching band. An exactly analogous result is seen in the SEIRA spectrum. If the analyte molecules were oriented on deposition in DD GC/SEIRA measurements, one would expect to observe significant difference between the SEIRA spectrum and the standard reference spectrum. Since we do not observe such differences, we must conclude that the molecules are randomly oriented. CONCLUSION Coating the ZnSe substrate that is used for DD GC/FT-IR measurements leads to a reduction in the limit at which molecules eluting from a gas chromatograph can be identified in comparison to any other GC/FT-IR technique. Provided that the silver film is sufficiently thin, distortions in the measured spectra are precluded and the analyte can be identified by library searching against standard databases of condensed-phase infrared spectra at levels that are equal to or lower than standard GC/MS interfaces and with better specificity for the identification of compositional isomers. ACKNOWLEDGMENT We thank Kathryn Kalasinsky of the Armed Forces Institute of Pathology for the generous loan of the Bourne Scientific IRC. Received for review April 6, 2005. Accepted July 10, 2005. AC050585L
