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Performance Characteristics of a Real-Time Direct Deposition Gas Chromatography/Fourier Transform Infrared Spectrometry System Sidney Bourne Bio-Rad Laboratories, Digilab Division, 237 Putnam Avenue, Cambridge, Massachusetts 02139
Andrew M. Haefner' and Kelly L. Norton Department of Chemistry, University of California, Riverside, Riverside, California 92521 Peter R. Griffiths* Department of Chemistry, University of Idaho, Moscow, Idaho 83843
I n this paper, the real-time measurmnt of the infrared speclraol rukrcmpqarqu"dandykrthathavebeen wpcurkd by gw dwenWog?aphy (QC) b &"strated for the fkst the. Ehltea trappod as small spots on a moving wk#kw hekl at 77 K ace past.d throwgh the beam of a Fowler transfarm Infrerd (Fl-IR) @"meter that Is 100 wn square. FlSaMme GWFT-IR spectra obtahed from InJectedquaniltkm as low as 50 pg are shown and the effect of pWrun signal-averaging In improving the signal-to-ndse ratio k demol#drated. With Hdr t e c w , subnanogram quantl#er of n-alkarm, at least up to , H C can be disqlm hated. 1d.nHRCatlon of polar, hydranslyles Is =-bYspbchd--wm-m* of wmpomb preparedasKBr dkkr. For krr pdar anadytes, spectralseMdrlng agskwt a databrrseal vaporphsse spectra Is also successful.
INTRODUCTION Three fundamentally different types of interfaces between a gas chromatograph and a Fourier transform infrared spectrometer can be distinguished. The most common incorporates a flow-through lightpipe gas cell with vapor-phase spectra being measured in real-time a t intervals of approximately 1 s (1-3). For systems of this type, the minimum identifiable quantity (MIQ) is rarely less than 5 ng, with the MIQ typically increasing by an order of magnitude for compounds of low absorptivity eluting from the GC column as broad peaks. The relatively low sensitivity of lightpipe-based GC/FT-IR interfaces has been addressed thtough the use of matrix isolation (MI) or direct deposition (DD) techniques. In GC/MI-FT-IR, each GC peak is trapped in a matrix of argon that is condensed on a moving metallic substrate, and the reflection-absorption spectrum is measured (3-6). In the most common GC/MI-FT-IR system, the Mattson Cryolect, the argon trace is about 300 pm in width, leading to an approximately 10-fold increase in absorbance from a given component in comparison to the corresponding GC peak measured with a 1-mm-diameterlightpipe (assumingthat the peak absorptivity of each band does not change, vide infra) (435).
In the DD interface, GC eluites are condensed on a cooled moving window without dilution in a matrix of any type. It 'Current address: Exxon, 5200 Bayway Dr., Baytown, TX 77522-5200. 0003-2700/90/0362-2448$02.50/0
has been demonstrated that the width of the trace, w,can be reduced to about 100 pm ( 7 , 8 )leading to an approximately 10-fold increase in the absorbance per unit weight over a GC/MI-FT-IR interface with a 300-pm-wide trace (again assuming no change in band absorptivities). Two GC/DDFT-IR interfaces have been described previously ( 7 , 9 ) . In the first, the window was thermoelectricallycooled to -35 "C. Although this temperature was too high to permit volatile compounds to condense on the window, it allowed operation in the sample chamber of a purged FT-IRmicroscope (7).The second unit, which operated below 100 K, was installed on the optical bench of an evacuable FT-IRspectrometer (9). A ZnSe window was cooled by circulating liquid nitrogen (LN,) through the brass window mount, requiring an excessively high flow of the cryogen to maintain the window a t its operating temperature. Results obtained by using both systems were promising, but subnanogram MIQs were never reported on either unit for measurements made in real-time. Decreasing the width of the trace for either a MI or DD measurement has three main advantages. (i) Since the noise equivalent power of an infrared detector is inversely proportional to the square root of the detector area, the signalto-noise ratio (SNR) of the spectnun is increased in proportion to w-l by reducing the dimensions of the detector element to match the width of the trace (10). (ii) For efficient optical configurations, the SNR of FT-IRmeasurements made using a narrow-band mercury cadmium telluride (MCT) detector can be limited by the dynamic range of the analog-to-digital converter (ADC), or the linear range of the detector or amplifier, even when the sample diameter is less than 0.3 mm. By reduction of the sample area (and hence the optical throughput) to the point that the SNR at the interferogram centerburst is less than or equal to the dynamic range of the ADC, the optimum spectral SNR can be obtained. (iii) With a decrease of the cross-sectional area of each eluite, its absorbance per unit weight is increased. Thus the deleterious effects of atmospheric interferences or poor baseline flatness, which limit the amount of ordinate expansion that can be achieved on spectra with otherwise high SNR, are minimized. In general, real benefits can be gained by reducing the width of the trace in any GC/FT-IR measurement involving eluite trapping to 100 pm. For w 5 50 pm, however, the practical difficulties of optical and mechanical alignment offset the theoretical benefits listed above. A second advantage that has been claimed for GC/MIFT-IR measurements in comparison to the corresponding measurements made with a lightpipe is the increase in peak absorptivity, since the full-width at half-height (fwhh) of bands @ 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990 0.124 A b 0.10
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Figure 1. Schematic of the optics and sample collection system for
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in MI spectra (typically 0.8-8 cm-’) is significantly less than the fwhh of corresponding bands in vapor-phase spectra (18 cm-’) and the integrated area is approximately constant (3, 5). Although the baseline noise level is always lowest for spectra measured a t low resolution in a given time, the increased absorbance of narrow bands measured a t high resolution partially offsets the increased noise level (IO). For real-time measurements, the acquisition time per spectrum must be kept below 1 s, and so in practice it is rare to find any real-time GC/FT-IR measurement made at higher resolution than 4 cm-’. Typical values for the fwhh of bands in GC/DD-FT-IR spectra fall between 4 and 8 cm-’; thus little difference in peak absorptivities should be observed between real-time MI or DD measurements made at 4-cm-l resolution. In practice, no GC/MI-FT-IR spectra have been measured in real-time, and all reported spectra of subnanogram sample quantities have been measured with postrun signal-averaging. The lowest quantity for which an identifiable spectrum has been obtained by GC/MI-FT-IR is 156 pg of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1234-TCDD), a strong infrared absorber, which necessitated signal-averaging 5000 scans (1I). To the knowledge of these authors, the only previous real-time G C / R measurement in which an MIQ of less than 500 pg has been reported was for 400 pg of 4-acetylmorpholine (an exceptionally strong infrared absorber) measured with a special lightpipe and an optical configuration for which few experimental details were given (1.2). rn this paper, we report a significant reduction in the MIQ for routine real-time GC/ FT-IR measurements and the first GC/IR spectra obtained from less than 100 pg of any analyte.
EXPERIMENTAL SECTION A schematic of the optics and sample collection system used to obtain the data in this paper (Tracer GC/FT-IR interface, Bio-Rad, Digilab Division, Cambridge, MA) is shown in Figure 1. The beam from a BieRad Digilab Model FTS-45 spectrometer enters the vacuum chamber through a potassium chloride window and is focused onto the sample window by an ellipsoidal mirror. The transmitted beam is collected by a Schwartzchildmicroscope objective, which projects an image of the sample through a field lens and an adjustable rectangular aperture to define the size of the image. A second Schwartzchild objective then focuses the beam onto a O.l-mm MCT detector. The eluites are deposited on a 60- X 30-mm ZnSe window mounted on a copper cold-block with a 50- X 25-mm rectangular cutout. The cold-block/window assembly is mounted on a motorized XY stage inside a vacuum chamber. The chamber is pumped with a 170 L/s turbomolecular pump, which maintains a pressure in the low Torr range with an inlet flow of 1-3 mL/min of helium. The cold-block is cooled with LN2 held in a 3-L Dewar with a flexible copper braid used for thermal contact
r
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Figure 2. ReaCtima GCIDDFT-IR spectra of (above)3 ng and (below) 160 pg of 3sthylphenol.
between the moving cold-block and the Dewar. The temperature of the window never exceeds 97 K. One charge of LN2keeps the ZnSe window at working temperature for about 30 h. For the separations reported in this paper, 1-pL samples were injected with a 301 or a 50:l inlet split ratio. Temperatureprogrammed separations were effected using a 30-m-long, 250pm-i.d. capillary GC column coated with a 0.25-pm-thick layer of cross-linked 5% phenyl 95% methyl polysiloxane. The initial helium flow rate was 0.9 cm3/min. A fused silica transfer line, butt-connected to the column inside the oven of a HewlettPackard Model 5890A gas chromatograph, delivers the sample to the window. To maintain a uniform temperature across the entire transfer line, two independently controlled heat zones were required: one for the main length of the transfer line from the GC to the cold-block and a second zone for the terminal 8 cm of the line which incorporatesan orifice/restrictor of 50-pm internal diameter. The output orifice is positioned about 50 pm from the surface of the window close to the location of the beam focus. Each eluite is deposited in an approximately elliptical area, with the dimensions being about 100 pm across the width of each eluite and 120 and 200 pm in the direction of movement of the window. The XY stage moves the window continuously throughout the chromatogram, so that each deposit& component passes through the beam several seconds after it is frozen on the window. In any chromatographic detector, there is always a delay between the moment a given eluite emerges from the column and the point it enters the detector. This time is usually on the order of 1 or 2 s. For the measurementsreported in this paper, the time delay is on the order of 15 s. Since the spectra are measured as each eluite passes through the infrared beam, the measurements can be described as being made in “real-time”. All spectra shown in this paper were measured at a resolution of 8 cm-’. The measurement time for the real-time spectra shown in this paper was 3 s or less. The position and movement of the window are controlled and recorded during a GC run by the spectrometer’s software. The window is translated in 25-pm increments at such a rate that it moves about 100 pm during the fwhh of a typical GC peak. The rate can be programmed to change during the course of the run to accommodate broadening GC peaks. At the end of the separation, it is possible to reposition the window so that a selected component is located in the optical beam for extended postrun signal averaging.
RESULTS AND DISCUSSION The temperature of the window was sufficiently low to retain even very volatile compounds. For example, no detectable loss of dichloromethane was observed after 4 h. The high sensitivity of real-time measurements is readily illustrated by the data in Figures 2-6. The spectra of 3 ng and 160 pg of 3-ethylphenol are shown in Figure 2, and spectra of 50 pg of isobutyl methacrylate, 3-ethylphenol, and n-dodecane are shown in Figures 3A, 4A, and 5A, respectively. These spectra were measured about 15 s after deposition of each eluite while the spot was moving through the infrared beam. The detection limits of GC/DD-FT-IR measurements can be further reduced by postrun signal-averaging, provided that the peak can be distinguished in the chromatogram. To demonstrate this capability, spectra of the same three 50-pg deposits of
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
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Figure 3. GC/DD-FT-IR spectra of 50 pg of isobutyl methacrylate measured (A) in real time and (B) by postrun signalaveraging. 3 3144
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Figure 4. GWDD-FT-IR spectra of 50 pg of ðylphenol measured (A) in real time and (B) by postrun signal-averaging. 0 064
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Figure 7. Functional group chromatogram constructed from the absorbance in the C-H stretching region for n-alkanes from n-CaH, Note that only those alkanes with even carbon through n-C,H,,. numbers are shown above n-C2&t,,.
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Flgure 5. GC/MTFT-IR spectra of 50 pg of ndodecane measured (A) in real time and (B) by postrun signal-averaging.
isobutyl methacrylate, 3-ethylphenol, and n-dodecane are shown in Figures 3B, 4B, and 5B after measurement with postrun signal averaging. Two hundred scans were averaged for each spectrum, corresponding to a data acquisition time of about 50 s. Although detection limits lower than this can only be obtained from molecules with large absorptivities and through the application of postrun signal-averagingfor longer than 1 min, it is usually a routine matter to measure the spectrum of 1 ng of even weakly absorbing compounds in real-time. Examples of real-time spectra of polycycljc aromatic hydrocarbons, which are notoriously weak absorbers, injected at the 1-ng level are shown in Figure 6.
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1.4 15 16 17 CARBON NUMBER
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Flgure 8. Plot of ratio of the peek absorbance of the antisymmetric stretching bands of the CH, and CH, groups of n-alkanes vs carbon number. Each alkane was injected at the level of 400 pg and the spectra were measured in real time.
The identification of simple compounds from their infrared spectra can sometimes be achieved by manual interpretation using Colthup-type correlation tables, but the higher the molecular weight of the molecule, the more difficult an accurate structural assignment. For example, distinguishing between straight-chain alkanes,n-CnHa+z,solely on the basis of their infrared spectra is difficult (13) and the difficulty increases with the carbon number, n. Nevertheless, it has long been recognized that the relative intensity of C-H stretching bands due to CH2 and CH3 groups increases with n. This property can be used to distinguish between n-alkanes using
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
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Figuro 8. Functional group chromatogram constructed from the absorbance In the C=O Stretching region for six barbiturates(barbital, aprobarbital, butabarbital, amobarbital, mephobarbital, and phenobarbital) injected at the level of 600 pg.
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GC/IR, even when subnanogram sample quantities are injected. The C-H functional group chromatogram of n - C a m through n-CMHU,each of which was injected at the level of 400 pg, is shown in Figure 7. The ratio of the peak absorbance of the antisymmetric C-H stretching bands of CH2groups (at -2918 cm-') and CH3 groups (at -2953 cm-') for these spectra increases smoothly with carbon number, as shown in Figure 8, indicating that homologues can be distinguished despite the weakness of these bands. The limiting carbon number appears to be determined by the SNR of the CH, band, which decreases as the carbon number increases. In these data, the first outlying point appeared at n-C24H50.It may, of course, be validly claimed that the n-alkanes are easily distinguished on the basis of their retention times. Certain branched alkanes exhibit similar retention characteristics to the n-alkanes, however, and the use of relative band intensities in the C-H stretching region can assist the identification of alkanes separated by GC. It is common practice to identify compounds for which GC/IR spectra have been obtained by spectral searching against the entries in a suitable database. The number of compounds for which either vapor-phase or matrix isolation reference spectra are available is small (100000). One of the principal advantages of GC/IR spectra measured by direct deposition is that they may be directly searched against libraries of KBr-disk reference spectra. To illustrate this capability, several barbiturates were separated; the carbonyl functional group chromatogram from 600 pg each of barbital, aprobarbital, butabarbital, amobarbital, mephobarbital, and phenobarbital is shown in Figure 9. Because barbiturates differ structurally by changes in just two substituents, the spectra of these compounds are similar, as shown in Figure 10. Even so, in most cases they can be identified by searching against the Georgia State Crime Laboratory (GSCL) Library of Infrared Spectra of Commonly Abused Drugs. For this search, the square of the derivative spectrum was used to guard against the effect of baseline slope. Spectra were searched in the region between 1800 and 700 cm-'. The results for aprobarbital are shown in Figure 11, and it can be seen that, even though differentiation between the first few hits is too low for certain identification in the absence of other information, the anal@ is correctly identified despite the similarity of the spectra of the barbiturates in the GSCL Library. Similarly, the other five barbiturates were also correctly identified, in each case as the first hit. Only when the analyte exists in several polymorphic forms does spectral searching in this manner break down (14), either because the sample used for the reference spectrum in the library has a different crystal structure than the deposit formed when the GC eluite condenses on the cold window or because the reference
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Figure 10. Real-time GC/DDFT-IR spectra of each of the barbiturate peaks shown in Figure 9.
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Flgure 11. Results of spectral searching the peak due to 6 ng of aprobarbital against the GSCL Library. The reference spectra for aprobarbltal yields a hit quality index (Euclidean dlstance) of 0.85 compared to 0.89 for the next best hit (butabarbital).
spectrum has been obtained from a mixture of polymorphs. Barbiturates contain strongly hydrogen bonding groups, so that their vapor-phase spectra are very different from their condensed-phase spectra and libraries such as the Environmental Protection Agency Library of Vapor-Phase Infrared Spectra (EPALIB) cannot be used for spectral searching. For compounds that do not contain hydrogen-bond donors, the intermolecular interactions are weaker and there are fewer differences between their vapor-phase and condensed-phase spectra. To illustrate this effect, the center wavenumbers of each of the major bands in the vapor-phase reference spectrum and direct-deposition GC/IR spectrum of the chlorinated pesticide, Aldrin, are listed in Table I. Below 2000 cm-' the average frequency shift for these bands is less than 3 cm-'. The similarity between these spectra allows Aldrin to be readily identified by spectral searching against EPALIB, as shown in Figure 12. The spectral shifts are higher in the C-H stretching region, with an average shift of 12 cm-'. This effect had been observed previously by Gomez-Tayloret aL ( E ) who , showed that this spectral region was sensitive to the local environment of several chlorinated insecticides. Spectral searching against a library of vapor-phase spectra may therefore give the best results for these compounds if only the
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
Table I. Center Wavenumbers of Main Bands in the Spectra of Aldrin Measured by Direct-Deposition GC/FT-IR and from the EPALIB Vapor-Phase Reference Library direct deposition
vapor phase
Au
3127 w 3059 m 3035 m 2975 s 2961 sh 2904 m
3131 w 3071 m 3055 m 2989 s 2974 sh 2916 m
4 12 20 14 13 12
1599 s 1469 m 1314 s 1249 s 1176 s 1107 m 1186 m 1067 m 1033 vs 932 sh 894 s 887 s 831 s 811 s 773 m
1598 s 1474 m 1319 s 1250 s 1174 s 1106 sh 1183 sh 1060 sh 1033 vs 932 sh 903 s 887 s 826 s 811 s 772 m
1 5 5 1 2 1
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2 epalib8113111 0.86 58-89-9
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ACKNOWLEDGMENT We wish to thank Senja Compton of Bio-Rad for performing many of the spectral searches and preparing some of the figures for this paper.
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where the reference materials are in a similar state to the unknown analyte. For direct-deposition GC/IR spectra, the analytes are in the solid state, indicating that the reference materials should have been prepared as either KBr disks or oil mulls. A more detailed study of the effect of sample preparation on spectral searching is underway (16),and results will be reported in the near future. In summary, we have demonstrated that the direct deposition of GC eluites as very small spots on a moving cooled window yields lower detection limits for GC/FT-IR measurements made either on-the-fly or after extended signalaveraging than any other technique in common usage. We have indicated that spectra measured in this way can be searched against conventional libraries of infrared spectra of reference materials prepared as KBr disks.
DEL ISOflER
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Flgure 12. Result of spectral searching the GC/DD-FT-IR spectrum obtained from 6 ng (inJected) of Aldrin (bottom trace) against the EPA Library of Vapor-Phase Infrared Spectra.
fingerprint region (2000-600 cm-') is included for the search. The stronger the intermolecular interactions, the greater the magnitude of the spectral shifts. When the analytes contain strong hydrogen-bond donors (such as the barbiturates), it is imperative to use a database for spectral searching
(1) Grifflhs, P. R.; de Haseth, J. A,; Ararraga, L. V. Anal. Chem. 1983, 55, 1361A-1371A. (2) Krishnan, K. Focuier Transform InParedSpecbpswpy; Ferraro, J. R., Easile, L. J., Eds.; Academic Press: New York, 1985: Vol. 4, Chapter 3. (3) White, R. ChromatognphylFowier Transform Infnrred S p e c t r o ~ y and its Applhtlons; Marcel Dekker: New York, 1990. (41 . . Bourne. S.;Reedy. 0. T.: Cunninaham, P. T. J . Chromatow. - Sci. 1970, 17. 460-463. (5) Reedy, G. T.; Bourne, S.; Cunningham, P. T. Anal. Chem. 1979, 51, 153511540. (6) Reedy, G. T.; Ettinger, D. G.; Schneider, J. F.; &me, S. Anal. Chem. 1085, 57, 1602-1609. ( 7 ) Fuoco, R.; Shafer, K. H.; Griffiths, P. R. Anal. Chem. 1988, 58, 3249-3254. ( 8 ) Griffiths. P. R.; Henry, D. E. Prog. Anal. Spectrosc. W88, 9 , 455-482. (9) Haefner, A. M.; Norton, K. L.; Griffiths. P. R.; Bourne, S.; Curbelo, R. Anal. Chem. 1980, 60, 2441-2444. (10) GriffRhs, P. R.; de Haseth, J. A. FwrkK T r a n s f m Inhgred Sp6cf"etty; Wiley-Interscience: New York, 1986 pp 248-259. (11) Holbway, T. T.; Fairkss, 8. J.; Friedline, C. E.; Klmball, H. J.; Kbepfer, R. D.; Wurrey, C. J.; Jonooby. L. A,; Palmer, H. G. Appl. Spectrosc. 1988. 42, 359-369. (12) HkSChfleld, T. Appl. S ~ ~ C ~ ~ 1085, O S C 39. . 1066-1087. (13) Sherman, J. W.; de Haseth, J. A,; Cameron, D. G. Appl. Spectrosc. 1989, 43, 1311-1316. (14) Haefner, A. M.; Norton, K. L.; Makshima, H.; Griffiths, P. R. Pittsburgh Conference on Analytlcal Chemlstry and Applled Spectroscopy, Atlanta, GA (March, 1989), Paper No. 459, 1969. (15) Gomez-Taylor, M. M.; Kuehl, D.;Grifflths, P. R. Appl. Spectrosc. 1978, 30, 447-452. (16) Norton, K. L.; Griffiths, P. R. Unpubbhed results, 1990.
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RECEIVED for review June 7,1990. Accepted August 17,1990. A.M.H., K.L.N., and P.R.G. wish to acknowledge partial support for this work by the US.Environmental Protection Agency under Grant R-814441-02 and Cooperative Agreement CR-812258-03.
