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Anal. Chem. 1989, 6 1 , 1678-1685
evanescent field sensing, this work has shown its usefulness in remote in situ process monitoring when specific chemistries are applied to the waveguide. The sensors provide an adequate signal-to-noise ratio and reproducibility with a fast response time (2 min after encountering the sample) for in situ measurements.
ACKNOWLEDGMENT We thank Lloyd Burgess for many helpful discussions during the course of this work. LITERATURE CITED (1) Jones, T. P.; Porter, M. D. Anal. Chem. 1888, 60, 404-406. (2) Kirkbright. 0.F.; Narayanaswamy,R.; Weiti, N. A. Analyst 1984, 109, 1025-1028. (3) Munkholm, C.; Wait, D. R.; Miianovich, F. P.; Klainer, S. M. Anal. Chem. 1888, 58, 1427-1430.
(4) Dawber, J. 0.:Wyatt, P. A. H. J . &em. Soc. 1960, 3589-3593. (5) Paul, M. A.; Long, F. A. Chem. Rev. 1857, 56. 1-45. (6) Rochester, C. H. AcMW functions: Academic Press: New York, 1970. (7) Hanick, N. J. Internal Reflection Spectroscopy; Interscience: New York. 1967. (8) Simhony, S.;Kosower, E. M.; Katzlr, A. Appl. Phys. Lett. 1888, 49, 253-254. (9) Paul. P. H.: Kychakoff,0. Appl. Phys. Lett. 1867, 51, 12-14. (IO) DeGrandpre, M. D.; Burgess, L. W. Anal. Chem. 1988, 6 0 , 2582-2586. (11) Seitz, W. R. Anal. Chem. 1884, 56. l6A-34A. (12) Manifacier, J. C.: Gasiot, J.; FUiard, J. P. J . Phys. E . 1876, 9. 1002- 1004. (13) Chedin, M. J. J . Chlm. Phys. 1852. 49, 109-125. (14) Smock, P. L.; Orofino, T. A.; Wooten, G. W.; Spencer, W. S. Anal. Chem. 1079, 57, 505-508. (15) Russell, A. P.; Fletcher, K. S. Anal. Chlm. Acta 1885, 170. 209-216.
RECEIVED for review March 14,1989. Accepted May 1,1989.
Picogram Level Quantitation of 2,3,7,8=Tetrachlorodibenzo=p=dioxin in Fish Extracts by Capillary Gas Chromatography/Matrix Isolation/Fourier Transform Infrared Spectrometry Magdi M. Mossoba,* Richard A. Niemann, and Jo-Yun T. Chen
Division of Contaminants Chemistry, Food and Drug Administration, 200 C St., S. W., Washington, D.C. 20204
For the fktt Ume, gas chromatography/matrbr kdetlon/fowler transform infrared spectrometry (GC/MI/FTIR) has been reported to confirm the Identity of 2,3,7,8-tetrachlorodibenzo-p-dloxin (2378-TCDD) and to quantify its level in fish extracts In the 170-220 pg range "on disk". When expressed on a fish tlssue basis, analyte levels ranged from 15 to 45 pg/g. Spectroscopic klentkatlon was based on the position and relative Intensity of seven absorption bands. Optlcai allgnment as well as performance evaluatkii and opthlzatbn of the GC/MI/FTIR system are descrlbed. The use of ['3C,2]2378-TCDD as an Internal standard was essential for quantltatlon, and quailty assurance controls were used to verify system performance. GC/MI/FTIR quantltatlon of 2378-TCDD was compared wlth that Independently found by GC wlth electron capture detectlon. Recovery of 2378-TCDD averaged 52% ( n = 8, 30% relative standard devlation) for fish extracts.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (2378-TCDD) (I) is an environmental contaminant that has received considerable attention. Concern over its extreme toxicity to laboratory test animals has required application of analytical methods at the pg/g (parts per trillion, pptr) level. Capillary gas chromatography/mass spectrometry (GC/MS) in the selected ion mode has been the preferred technique for measuring dioxins because of high sensitivity and structure specificity, but dioxin isomers generally cannot be distinguished by electron impact GC/MS. Capillary GC/Fourier transform infrared spectrometry (GC/FTIR) logically complements GC/MS by providing information on isomer identity (2). However, onthe-fly GC/FTIR has inadequate sensitivity for 2378-TCDD
residue application. The GC/matrix isolation/FTIR technique (GC/MI/FTIR) ( 3 , 4 ) has greater sensitivity, since the chromatographicallyseparated components are trapped at 12 K in a microscopic argon matrix, thus allowing an increase in the FTIR spectral acquisition time. Because each analyte molecule is isolated in a cryogenic solid matrix of IR-transparent argon atoms, a decrease in the absorption bandwidths may be observed, leading to an increase in band absorbances. GC/MI/FTIR instrumentation has been used to obtain spectra of the 22 TCDD isomers (561,the 13C12-labeledand the unlabeled (12C) 2378-TCDD (7), but these studies were limited to standards and/or to analyte levels one or more orders of magnitude greater than would normally be isolated from a biological matrix such as fish. Unequivocal spectroscopic identification of compounds at subnanogram levels has already been achieved by GC/MI/ FTIR (4, 7,8).The observed positions and relative intensities of FTIR bands allowed spectral interpretation at these levels. We have used GC/MI/FTIR to confirm the identity of 2378-TCDD in fish extracts and to quantitate it at the picogram level. In this paper we describe procedures for optimization of detectability and calibration and the use of an isotopically labeled ['3CrZ] 237&TCDD as an internal standard. We have also identified several extract components that are potential interferences.
EXPERIMENTAL SECTION Gas Chromatography and Cryolect Interface. Gas chromatographic separations were performed on a Hewlett-Packard Model 5890 equipped with a BaNielectron capture (EC)detector and connected to a Hewlett-Packard 3392A integrator. A 25 m X 0.20 mm i.d. cross-linked methyl silicone capillary column, 0.33 p m stationary phase (Hewlett-Packard),was used throughout this study. Helium containing 1.5% argon (Matheson Gas Products)
This article not subject to U S . Copyright. Published 1989 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
at approximately 27 cm/s linear velocity was used as carrier gas, and nitrogen (99.999%) at 45 mL/min was used as the make-up gas to the EC detector. The injector and detector temperatures were 250 and 350 "C, respectively. The carrier and make-up gases were passed through filters which removed traces of moisture (Alltech Associates Hydro-Purge 11) and oxygen (Supelco Gas Purifier and MG Scientific Gases Oxisorb, for He/Ar and N,, respectively). Splitless injections of 1-3 pL of isooctane solutions were made over 10-15 s, and the injector was purged at 2.75 min. The initial column oven temperature was 75 "C with a 2-min hold, followed by a 20 "C/min increase to 270 "C, and the column oven was held at this temperature until the separation was complete. The Cryolect interface (9) as supplied (Mattson Instruments, Madison, WI) required some modification and considerable optimization. The end of the capillary column exited through the top of the oven and was connected to one end of a union (SGE, Austin, TX) attached to a small, cartridge-heated block placed on the top oven wall insulation. At the other end of the union the effluent was split. Two splitter unions were tested. The fist was Model VSOS (SGE), a stock item, which had expcsed stainless steel at the conical end where the effluent is split. The second was a specially fabricated all-glass-lined (silanized deactivated) union (SGE) which was found to be more satisfactory in preserving TCDD peak shape. The column effluent was split to two pieces of fused silica tubing, each 0.7 m long: a 0.17 mm i.d. piece went back into the column oven and was inserted into the EC detector, while a 0.20 mm i.d. piece was connected to an open-split interface (Valco cross) located on top of the heated block. Connected to the opposite port of the cross was the 0.17 mm i.d. X 1m heated (270 "C) fused silica transfer line to the evacuated chamber (lo4 Torr) in which the cryogenic (11-12 K) collection disk was located. The remaining two perpendicular cross ports served as a pneumatic switch. Whenever deposition on disk was not required, purified helium swept across these two other ports at 60 mL/min to vent the column effluent away from the transfer line going to the collection disk. During the deposition cycle, the helium flow was cut back to 0.1 mL/min, which allowed carrier gas from a line that is T-connected to the vent line to flow into the cross as a make-up between the total flow through the collection disk transfer line (I mL/min) and the column flow into the cross (0.9 mL/min). Since column flow rate was nearly equal to the maximum flow through the transfer line, loss of analyte was avoided by setting the carrier gas make-up flow rate to half its recommended value, i.e., to 2.5 mL/min. Peak absorbance measured on disk then increased 20%. The splitter union, splitter lines, and open-split interface were all covered with insulation, and the block was heated to 270 "C. The column effluent split ratio was calculated from EC response factors (area counts per nanogram injected) obtained with 10-ng injections of 1234-TCDD made with the column attached to the splitter union, and again with the column outlet directly inserted into the EC detector. A ratio of 99.5:0.5 was so determined, with the smaller portion going to the EC detector. MI/FTIR Instrumentation. A Mattson Instruments Sirius 100 FT-IR spectrometer equipped with a Cryolect matrix isolation unit was used. The Cryolect system has been described in detail (3,4,9),except for the optical bench alignment procedure, which will be discussed here. The IR beam from the spectrometer is focused onto the frozen argon matrix by the first mirror of an independently adjustable double parabolic mirror assembly. The gold-plated mirror surface of the collection disk reflects the transmitted light back through the matrix to the second mirror of the assembly. The light then travels to a thud parabolic mirror which focuses the beam onto the element surface area, 760 X 760 pm2,of a narrow band (750 cm-' cutoff), high sensitivity mercury cadmium telluride (MCT) detector cooled to liquid nitrogen temperature. The IR beam must be focused on the cryogenic disk as a microscopic circular dot which samples the analyte preserved in frozen argon. Otherwise, an optically distorted beam will degrade the spectroscopic signal and the IR-reconstructed chromatographic peak shape. The Cryolect optical bench alignment used an external helium-neon gas laser mounted along the spectrometer output light scribe line. To align the spectrometer and the Cryolect optical benches, a flat mirror was placed on the Cryolect bench and its position adjusted between the second and third parabolic mirrors
1679
along the light path until the reflected laser light fell on the laser source aperture. Next the position of each of the two mirrors of the double parabolic mirror assembly was independently adjusted so that the incident and reflected laser beams were visible at only one spot on the gold-plated collection disk. The external laser was then removed, and a quartz beam splitter was substituted for the KBr beam splitter. By use of two alignment stands placed on the spectrometer output beam scribe line, the position of the flat mirror that diverts the beam toward the Cryolect bench was optimized. With the iris set at its smallest aperture, the focus of each of the two parabolic mirrors of the assembly was further adjusted so that the incident circular image of the source visually overlaid the reflected image on the collection disk. After the KBr beam splitter was reinstalled and the flat mirror on the Cryolect bench was removed, the second and third parabolic mirrors were successively adjusted until the interferogram centerburst was maximized. The vacuum chamber was pumped down and the disk cooled to 11K. By use of a quartz beam splitter again, the height of the double parabolic mirrors assembly unit was visually adjusted such that the beam is focused onto a 343-pm diameter reference notch drilled (number 80 drill bit, 0.0135 in. (0.343 mm)) into the collection disk; the height of the deposition tip of the transfer l i e had also been adjusted to that of the reference notch. The KBr beam splitter was reinstalled and the interferogram was remaximized, giving 12.7 V at 5% iris aperture. Fine tuning adjustments were made to the double parabolic mirror assembly by monitoring the major band absorbance of 8-10 ng of 1234TCDD on disk. It was observed that a 5% iris yielded a slightly higher absorbance value than did other apertures, which implied that the source image at the disk was probably optimally matching the size of the frozen analyte. Standards and Quality Assurance Solutions. All solutions were prepared in isooctane (Burdick & Jackson, distilled in glass). A 7.9 ng/pL solution of 1234-TCDD was used to optimize instrument performance. A 500 pg/pL working stock solution of 2378-TCDD was prepared from a concentrated standard (KOR Isotopes, Cambridge, MA). An aliquot of the working stock solution was used to prepare a 4.0 pg/pL fortification standard. A 15.0 pg/pL l3Cl2-1abeled2378-TCDD (National Institute of Standards and Technology SRM 1614) internal standard was prepared in a 5-mL volumetric flask by transferring and diluting the contents of a weighed ampule containing 96.6 ng of internal standard per gram of solution. The contents of a second weighed ampule of [l3Cl2]2378-TCDDwere transferred to a concentrator tube, the lower 1-mL portion of which was calibrated in 100-pL increments, and concentrated to 100 pL under a stream of purified nitrogen. Aliquots (25 or 63 pL; digital Micro/pettors, SMI, Emeryville, CA) from this tube were mixed with aliquots (40 or 100 pL) from the unlabeled working stock solution in two other tubes and were diluted to 200 pL to prepare two GC/MI/FTIR calibration standards. These two 2378-TCDD calibration standards contained '2c at 100 pg/pL + 13Cat 93 pg/pL, and 12C at 250 pg/pL + 13C at 234 pg/pL, respectively. The quality assurance (QA) solutions were prepared at various concentrations from aliquots of the unlabeled fortification standards and the internal standard. Test Sample Preparation. The edible portion of ground fish was analyzed by the method of Niemann et al. (IO)which includes base/acid treatment and off-line size exclusion, C-8, and (2-18 HPLC cleanup to isolate the analyte 2378-TCDD. The chosen fish had a bioincurred analyte level that had previously been determined by GC/MS with multiple ion detection. Although major contaminants in the cleaned-up extracts, which were materials bleeding off the C-18 columns, had not interfered with the MS-monitored ions, they did interfere severely with infrared identification of analyte. However, these components were completely removed by a Hamilton PRP-1 column (polystyrene gel, 5 pm in diameter, 4.6 mm i.d. X 15 cm, 100% AN at 1.0 mL/min, 45 "C) added as another stage of HPLC cleanup after that of the C-18 column. The collected dioxin fraction eluted shortly after the interfering material which came off at or near the void volume. After evaporation to dryness and residue uptake into 100 pL of isooctane, the analyte level was quantitated by fused silica capillary GC/EC under the following conditions: 0.2 mm i.d. X 50 m ULTRA No. 1 (bonded phase methyl silicone, Hewlett-Packard); temperature programmed at 20 "C/min from 75
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 15. AUGUST 1, 1989
A. Cryogenic Disk
Deposition Tip
yi
20 10
0 '4 4
148
46 PPtenlOn
6.
15 D
15 2
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Figure 2. IR-reconstructed GC peak shapes of 1234-TCDD standard on cryogenic disk obtained by monitoring the most intense band at 1433 cm-' and using a VSOS SGE splltter union: (A) before optical alignment; (B) after optical alignment; and (C) using a specially fa-
-I
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. . . . ,. , Matrix . 500 ptn
-
,
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Flgue 1. (A) Duhrg effluent trapping, the heated (270 "C)GC transfer line tip is about 100 pm from the slowly rotating cryogenic (12 K) collection disk located in a vacuum chamber. The solid surface in the y - r pkas is the cross sectbnal area of the frozen anatyte in the argon matrix. (B) After the GC separation is complete, FTIR measurement begins. The shape of ttw cross section of the solid analyte in the x-y plane is given as absorbance (y axis) vs GC retention time ( x axis) in
Figure 2 (see text). "C (2 min) to 250 "C (hold);hydrogen carrier gas at 40 cm/s (hear velocity measured at 250 "C); 300 "C injector; 350 "C detector; splitless injections of 1-pL solutions. Next, [l9C]2378-TCDD internal standard was added at 1-1.5 times the unlabeled level as screened by GC/EC, and the extract was submitted along with QA solutions for GC/MI/FTIR analysis. GC/MI/FTIR Analysis. The prepared extract was transferred to a sample tube (bottom part graduated in 10 & Kontes, Vineland, NJ) with two 0.1-mL isooctane rinses and concentrated under a stream of nitrogen while the tube was warmed in a water bath. When the volume reached about 20 rL, the tube walls were rinsed with 25 p L of isooctane and concentration was continued to about 3 ML. The solution was never allowed to go to dryness and was analyzed within a few hours. The entire volume (equivalent to 15 h 0.5 g of fish in the test portion), or as much as could be taken up into the syringe (1-3 pL), was injected. A typical day's run included two calibration standards, one or more QA solutions, and a fish extract. A; strongest Calibration plots of absorbance (in units of band) vs picograms injected were made for native and labeled analytes generated over 9 days of injecting calibration standards. The amount of 2378-TCDD present in test solutions (extracts or QA) was calculated from the observed milliabsorbance values, the 12Cand I3C regression line equations describing the calibration plots, and the picogram amounts of internal standard added. QA solution measurements were used to verify that the calibration curves had not shifted. At the start of a GC/MI/FTIR run, the Cryolect collection disk begins to rotate slowly at a predetermined speed (50 pm/s) and the effluent is directed toward the outer rim of this cryogenic collection disk, thus freezing instantly into a solid matrix (Figure 1). A cross section of the frozen analyte in the IR-transparent argon matrix, defined as beiig in the x-y plane, is shown in Figure 1B. By this definition, the disk moves along the x direction during effluent collection, while the thickness of the frozen analyte varies along they axis. Since the disk rotation and GC separation occur simultaneously during analyte collection, the distance along the x axis is indexed by the GC retention time. Thus, the position of a separated component trapped on disk is denoted by the
bricated allglass-lined union (SGE)after optical alignment (intensity normalized to 8.0 ng injection: retention time normalized to 14.7 min). Less tailing was found in (C) and the observed peak height, which served as a reference, exhibited increased intensity. retention time of its corresponding GC peak. The shape of the cross section of the frozen analyte in the x-y plane, as measured by FTIR, is the IR-reconstructed chromatographic peak shape. Hence, the independent variable ( x axis) is GC retention time and the dependent variable Cy axis) is absorbance (Figure 2; see Results and Discussion). The chromatographic retention time was used only to index the position on the collection disk of a component in the argon matrix to within a few seconds (up to several hundred micrometers). However, a rigorous Cryolect disk-scanning protocol was followed in order to reproducibily pinpoint the location of a peak maximum. For several consecutive measurements, when the computer-controlled stepper motor, " which rotates the collection disk, positioned a component (of known retention time) trapped in the argon matrix in the path of the IR beam, the observed absorbance for that component was different (up to 20%) each time. A backlash of at least 338 steps or 50 pm (1 s on the retention time scale) in the collection disk drive train appeared to prevent reproducible disk positioning under normal operating procedures. To overcome this problem, at 1.2-9 intervals we sampled a narrow window of about 5 s where we expected to find the maximum amount of analyte (top of GC peak). In this case the observed absorbance increased to its maximum value and then declined. The spectrum with the maximum absorbance is the one that corresponds to the configuration in which the focused IR beam optimally falls within the analyte area. Using this protocol, 1000 analyte interferograms were coadded (9 min 5 s at 4-cm-' resolution) at each sampling interval. The background was usually collected (lo00 scans) before or after the analyte peak.
RESULTS AND DISCUSSION The matrix isolation FTIR spectrum of 2378-TCDD (Figure 3A) exhibited about a dozen bands, with a maximum absorbance a t 1471 cm-'. Figure 3B shows the spectrum of [ l3CI2]2378-TCDD. Because the level of the 2378-TCDD isomer was in the subnanogram range, we first had to document and optimize the performance of the GC/MI/FTIR system to be able to confirm spectroscopically the identity of this relatively weak infrared absorber and quantitate its level in the various matrices. GC/MI/FTIR System Optimization. The IR-reconstructed chromatographic peak shape of the analyte on the Cryolect disk is a useful diagnostic tool which was used to monitor and improve the performance of both the GC interface and the Cryolect. The most intense band at 1433 cm-' of the standard 1234-TCDD was monitored for this purpose. The retention time of the IR-reconstructed chromatographic peak was always slightly shorter than the retention time observed by EC, since after the effluent is split, it follows two
ANIALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
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1400
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1100
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0 900
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Flgure 3. Observed MI/FTIR spectra in the 1600-800-~m-~ region acquired at 4-cm-' resolution by coadding 1000 scans (9 min 5 s) of (A) about 1.3 ng of 2378-TCDD standard injected and (B) about 1.1 ng of [I3C]2378-TCDD standard injected.
unequal pathways. The EC line is under pressure, whereas the Cryolect line flow is determined by the Cryolect chamber vacuum. This difference, which could be minimized, also depends on the nature of the GC interface (e.g., transfer lines lengths and internal diameters). Effects of Independently Adjustable Parabolic Mirrors and Laser Optical Alignment. The observed shape of the IR-reconstructed chromatographic peak (Figure 2) depended not only on the efficiency of the GC separation and the transfer process but also on the alignment and focus of the IR beam. When the mirrors of the parabolic mirror assembly were mounted in one fixed position, a distorted peak shape was obtained (Figure 2A). The cross-sectional area of such a beam appeared as a small circular area of condensed light as well as a less intense elongated coma when visible light was used. The shoulder on the leading edge of the IR-reconstructed peak was eliminated by replacing the fixed-position parabolic mirror assembly with one in which each mirror of the parabolic mirrors pair was replaced with an independently adjustable mirror that allowed proper alignment of the IR beam. This resulted in sharper peaks (Figure 2B,C). Effect of Splitter Union. The effect of splitter unions on the shape of the IR-reconstructed GC peak could then be determined and the peaks compared to those found by EC. When the original VSOS SGE splitter union was used, the IR-reconstructed GC peak height (Figure 2B) was less intense and showed more tailing (full width at half maximum, fwhm 5.4 s) than when a specially fabricated all-glass-lined SGE union was used, fwhm 5.2 s (Figure 2C), which justified our decision to use the all-glass-lined union. In the absence of any union, when the column was directly inserted into the EC detector, the chromatographic efficiency was good and a symmetrical peak shape was observed, fwhm 4.0 s (Figure 4A). The effect of using the original VSOS SGE splitter union is
~
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14.55
H 1 Min
Flgure 4. Partial GC/EC chromatograms. (A) 1234-TCDD standard, 7.9 ng injected, observed with the capillary column directly connected to EC, shows a symmetrical peak, fwhm 4.0 s; retention time 14.60 min; chart speed 0.6 cm/min. (B) A chlorinated pesticide standard mix shows no tailing with the original VSOS SGE union installed, fwhm 5.1 s for the middle peak (divert-to-vent mode). Mix contains dieldrin, heptachlor, heptachlor epoxide, DDE, and chlorpyrofos; elution order undetermined; levels range from 4 to 7 nglcomponent; chart speed 2.0 cm/min. (C) 1234-TCDD standard, 0.79 ng injected shows tailing and reduced peak height with the original VSOS SGE union installed; chart speed 2.0 cm/min. (D)Same as (C) using the specially fabricated allglass-lined SGE union, fwhm 5.7 s (dhrert-to-vent mode). The same EC response factor was found for both (C) and (D).
shown in Figure 4B,C. It is noted that pesticides (Figure 4B), but not TCDD (Figure 4C), produced symmetric GC peaks with no tailing. The GC/EC fwhm values observed with the special all-glass-lined SGE union were 5.7 s, when the flow was diverted to the vent port (Figure 4D), and 7.5 s, when the effluent was deposited onto the cryogenic disk. These results indicate that the extent of postcolumn broadening is smaller in the Cryolect (fwhm 5.2 s) than in the EC line (fwhm 5.7 s), and that the efficiency is only slightly worse than in the absence of unions (fwhm 4.0 s). Bourne and Croasmun (9) recently reported a larger fwhm value, 6.8 s, observed by FTIR under similar Cryolect conditions. Collection Disk Rotation Parameter. The IR-reconstructed GC peak shape also depends on the rotation speed of the collection disk, which is another independent factor that determines the physical length in micrometers over which an eluting compound will be spread. In general, this speed should be set so that an eluting component will not be deposited over a previously eluting component as long as their adjacent GC
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peaks are chromatographically resolved at the base line. The only available disk rotation speed value, 50 pm/s, was satisfactory. The distance along the collection disk surface that corresponds to the fwhm of the IR-reconstructed GC peak (5.2 s) is 260 pm, whereas the peak base width is close to 500 pm, excluding the tail. This is actually the length along the x axis of the cross section of the frozen analyte in the x-y plane (Figure 1B). The dimension along the z direction of the cross section of the solid analyte in the y-z plane (Figure 1A) could not be documented. Reedy et al. ( 4 ) pointed out that absorbance is inversely proportional to the square of the diameter of the area over which analytes are condensed and that the focused IR beam must fall within the surface area on which the analyte is collected. IR Beam Size on Disk. When the focused IR beam was allowed to fall onto the 343 pm i.d. reference notch located on the collection disk surface, the interferogram centerburst voltage was at a minimum of 2.1 V, which is about 17% of its maximum value of 12.7 V obtained with the beam focused on the disk mirror surface. This result indicated that the diameter of the focused IR beam cross-sectional surface area on the collector disk ( d I R ) is slightly (9.3%) over 343 pm. It is not known how dIR,which is 375 pm, compares with the width of the argon matrix, which is also the dimension of the cross section of the frozen analyte along the z direction of the deposition surface (Figure 1A). If such distance were smaller than 375 pm, an FTIR microscope would be needed for the IR beam to fall within the surface area of a deposited analyte. The use of a matching MCT detector element surface area is also a requirement. FTIR Characterizationof Standards. Several characteristic FTIR bands have been observed for the symmetrical and planar 2378-TCDD isomer (Figures 3A and 5B). Identification was based on the most intense seven bands: the 1575,1489, and 1471 cm-' absorbances arising from the C = C skeletal in-plane vibrations of the aromatic ring, the C-0-C asymmetric stretch a t 1312 cm-l, the aromatic ring trigonal bending at 1176 cm-l, the aromatic ring breathing at 1117 cm-', and the C--H out-of-plane bending a t 879 cm-'. In general, these absorption band positions are in agreement with those found for 2378-TCDD in KBr by Chen (11);however, band intensities are diminished, relative to the most intense 1471-cm-l band, in the argon matrix spectrum. Variations in band intensities for small planar molecules in matrix environments have been explained in terms of molecular orientation effects (12). With the exception of the C - 0 4 asymmetric stretch vibration, which appeared at the same frequency by both KBr and matrix isolation techniques, the other bands are found to have shifted to higher frequencies by 3-9 cm-' in the argon matrix. The bands observed by matrix isolation for the 13C-labeled2378-TCDD (Figures 3B and 5B) are shifted with respect to the unlabeled compound to lower frequencies by about 20-50 cm-l, and the separation between the two most intense bands used for quantitation (1471 cm-', lZC,and 1432 cm-', I3C) was 39 cm-'. Because the overlap between the spectra of the unlabeled and the labeled compounds was small, the [ 13C12]2378-TCDDwas used as an internal standard which was added to the prepared extract before GC/MI/FTIR analysis. The unlabeled and labeled 2378-TCDD isomers coelute, and thus are sprayed and condensed on the cryogenic collection disk at the same microscopic spot. The effect of signal averaging can be seen in the spectra of a standard solution containing 190 pg ("%) and 176 pg (13C) obtained after coadding 32-scan (Figure 5A) and 1000-scan (Figure 5B) interferograms of analyte and background. The seven bands used for the identification of the 2378-TCDD isomer could be observed satisfactorily, and signal averaging
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Figure 5. MI/FTIR spectra (4 cm-') observed for a standard solution containing 190 and 176 pg of ['*C]- and [13C]2378-TCDD, respectively, acquired by coadding (A) 32 scans (17 s) and (e) 1000 scans (9 min 5 s). Arrows indicate monitored bands for confirming the presence of 2378-TCDD (frequencies (cm-'): 1575, 1489, 1471, 1312, 1176, 1117, 879).
-0.005 Y
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Figwe 6. MI/FTIR spectrum of 2378-TCDD standard showing a small range around the most intense 1471-cm-' band; see text.
for times longer than 9 min 5 s (lo00 scans) was not necessary. The band intensities were obtained from the band heights measured on the plotted spectra. The heights of bands that overlapped with adjacent bands, such as the most intense two bands at 1471 and 1432 cm-l, were determined indirectly. From the spectrum obtained for either the 12Cor 13C alone (Figure 3), the constant ratio (AC/AB) could be found (Figure 6). For I2C and 13C mixtures, the distance AC could still be observed, and AB was calculated as the observed AC value divided by the known ratio (AC/AB). Fringes in the base line were often found in such low-level spectra and were probably due to multiple reflections of the IR beam between the col-
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Table I. Confirming the Identity of 2378-TCDD by Normalizing the Absorbance of Six Selected Characteristic IR Bands to the Absorbance of the Strongest Band at 1471 cm-' test material '*C standard (1.3 ng) carp 1,Oextract 224 carp 1,O extract 226 carp 1, extract 55 carp 1, extract 65 catfish, extract 146 carp 1, extract 68b carp 2, extract 77
carnivores composite, extract 69 acceptable absorbance ratio range: %
1575 cm-' 18 20 15 15 16 15
-
16 -b
16-21
absorbance ratio, %, at 1176 cm-'
1489 cm-'
1312 cm-'
31 38 28 35 30 31 25 34 24-36
29 24 33 22 22 24 23
1117 cm-'
13 14 15
879 cm-'
18 21 17 17 19 18
15 13 12
-
16
33 34 33 33 37 31 32
-b
-b
-b
-b
19-31
7.6-18
14-20
29-38
-b
"Fortified with ['*C]2378-TCDD. bNoisy. cRange = range of average ratios of absorbance at specified wavenumbers to that at 1471 cm-', as determined from five injections of calibration standards at amounts between 200 and 400 pg (1471 cm-' = 100%). lection disk mirror surface and the outer surface of the argon matrix. The background was usually collected close to the peak of interest, specifically about 12 s before or after the 14.77-min retention time of 2378-TCDD, in order to minimize the effects of the interference fringes. Analysis of Fish Extracts. Prepared fish extracts contained interferences in the IR measurements which were undetected by EC. These materials gave very intense FTIR bands consistent with a siloxane structure a t 2969 and 2935 cm-' (C-H stretch), 1021 cm-' ( S i 4 stretch), 1262 cm-' (CH3 in-plane bend), and 808 cm-' (CH3 rock), and obscured any TCDD bands that were present a t levels that were about 2 orders of magnitude lower than those of the contaminants. From earlier mass spectrometry work (10) we know that these interfering materials bled from the Du Pont C-18 HPLC columns used in the last step of extract cleanup. The structure of one component was recently identified (13)as bleeding from a Waters C-18 column. Fortunately, the bleed material was completely removed with a PRP-1 (Hamilton) column and the spectra of 2378-TCDD in fish extracts could be observed (Figure 7). The spectra of fish extracts (Figure 7) show the monitored analyte bands riding on patterns attributed to fringes. For extracts 226 and 55 derived from the same test portion, the fact that equivalent amounts of analyte were measured on disk even though one extract was prepared from fortified tissue, illustrates the highly variable efficiency of analyte removal from these tubes. The peak-to-peak noise level found for extract 226, containing 212 pg of 2378-TCDD "on disk", was 0.27 mAbs in the range 1600-1300 cm-' where the most intense band (1471 cm-', 4.6 mAbs) was found, yielding a signal-tonoise ratio (SNR) of 17:l. The MI/FTIR absorption bands can be sharp; however, this is evident only at resolutions that are high enough (e.g., 2 cm-' or 1cm-') to match the natural bandwidths of the absorption bands. While sharp bands can improve detectability and quantitation, data acquisition a t a higher resolution would require a measurement time that is much longer than the one used, 9 min 5 s, in order to recover the same SNR, 17:1, obtained at 4 cm-l. Identification of 2378-TCDD required satisfying the following criteria. The 12Cand 13C spectra were coincident a t the expected retention time on disk. The monitored seven characteristic bands of [12C]2378-TCDDand the major band of [13C]237&TCDDoccurred at the correct wavenumbers. The ratios of absorbances of the six weaker bands to the strongest one at 1471 cm-' (referenced as 100%) for a nanogram-level [12C]2378-TCDD standard (Table I) were similar to those respective ratios observed for calibration standards at equivalent or bracketing amounts expected from test analyses. Ratios were deemed similar if they fell within the range determined by the average f 2 standard deviations as defined
1
B
-0.012
-0.024
1i
1
I
I
I
l
I
I
I
900
800
11mAbs I
-1
I
1500
I
1400 1300
1200 1100 loo0 Wavenumber
,
Figure 7. MVFTIR spectra (1000 scans, 4 cm-') obtained at 2378TCDD retention time on disk for fish extracts: (A) extract number 226, a test portion of carp fortified at 55 pg/g (unlabeled), 212 pg "on disk" at 27% internal standard (IS) recovery; (B) extract number 55, same test portion of carp as (A) but unfortified, 221 pg "on disk" at 6 3 % IS recovery. Arrows indicate monitored bands for confirming the presence of 2378-TCDD (frequencies (cm-'): 1575, 1489, 1471, 1312, 1176, 1117, 879).
by standards. When all band ratios were outside the acceptable range, quantitation based upon the 1471-cm-' band was unreliable, and the presence of 2378-TCDD would not be considered as confirmed. When only one or two bands were outside the range by 1-2%, analyst judgment was exercised and the identity of 2378-TCDD might be confirmed. Table I presents the absorbance ratios and acceptable ranges for judging confirmation. The identity of 2378-TCDD could not be confirmed for fish extract 68, one of triplicate analyses of carp 1, because the spectrum was inexplicably noisy, and for extract 69, the lowest level extract (
