(12) R. Druckery, S. Preussmann, D. Ivankovic and 2. Schmahl, Krebsforsch.. 69, 103 (1967). (13) M. F. Argus and C. Hoch-Ligeti, J . Natl. Cancer Inst., 27, 695 (1961). (14) H. Druckery, R. Preussman, D. Schmahl, and M. Miller, Naturwissenscbaften, 48, 134 (1961). (15) E. Boyland, R. L. Carter, J. W. Gorrod, and F. J. C. Roe, Eur. J . Cancer, 4, 233 (1968). (16) H. Lund, Acta Chem. Scand., 11, 990 (1957). (17) L. Holleck and R . Schindler, Z . Nectrochem., 62, 942 (1958). (18) "Official Methods of Analysis", 12th ed., Association of Official Analytical Chemistry Washington, D.C., 1975. (19) R . A. Nicholas, and J. B. Fox, Jr., J . Assoc. Off. Anal. Chem., 56, 922 (1973). (20) S . S.Mirvish, J. Sams, T. Y. Fan, and S. R. Tannenbaum, J . Natl. Cancer Inst., 51, 1833 (1973). (21) S. Mirvish, Toxicol. Appl. Pharmacol., 31, 325 (1975). (22) S. Mirvish, J . Natl. Cancer Inst., 44, 633 (1970). (23) J. H. Ridd, Quart. Rev., Chem. Soc., 15, 418 (1961). (24) E. Kalatzis and J. H. Ridd. J . Chem. SOC.6 ,529 (1966). (25) T. Y . Fan and S. R. Tannenbaum, J . Agric. FocdChem., 21, 237 (1973). (26) C. A. Bunton, D. R. Llewellyn, and G. Stedman, J . Chem Soc., 568 (1959). (27) R. K. Skogerboe and C. L. Grant, Spectrosc. Lett., 3, 215 (1970). (28) J. 0 . Ingle, Jr., J . Chem. Educ., 51, 101 (1974). (29) S. R. Tannenbaum, A. J. Sinskey, M. Weisman, and W. Bishop, J . Natl. Cancer Inst.. 53, 79 (1974). (30) P. N. Magee, R. Montesano, and R. Pruessmann in "Chemical Carcinogens," ACS Monograph, No. 173, C. E. Searle, Ed., 1976, Chapter 11.
Table VII. Concentration ( p p m ) of NANO, in Frankfurter: Comparison of 1 : l O vs. 1:lo0 Dilution Concentration ( p p m ) o f NANO, in frankfurter 1:lOO ( n = 4 ) 1 : l O (n = 4) Sample D 23.44 i 0.10 23.56 i 0.28 Sample E 25.44 * 0.09 25.49 i 0.24 ACKNOWLEDGMENT T h e authors thank Walter Fiddler of T h e Eastern Regional Research Laboratories of the U S . Department of Agriculture for his generous contribution of materials for Griess reagent. LITERATURE CITED P. N. Magee and J. M. Barnes, Brit. J . Cancer, 10, 11 (1956). W. Lijinsky and S. S. Epstein, Nature (London), 225, 21 (1970) I. A. Wolff and A. E. Wasserman, Science, 177, 15 (1972). E. Szekely, Talanra 15, 795 (1968). E. Sawicki, T. W. Stanley, I. F-faff, and A. Damico, Tabnta, 10, 641 (1963). Princeton Applied Research Application Brief N-1, Princeton, N.J., 1974. S. W. Boese, V. S. Archer, and J. W. O'Laughlin. Anal. Chem.. 49. 479 (1977). S. Yanaqida, D. J. Barsotti, G. W. Harrinqton, and D. Swern. Tetrahedon Lett., 28, 2671 (1973). S. K. Chang and G. W. Harrington, Anal. Chem., 47, 1857 (1975). S. E. Abanoli, J. A. Popp, S. K . Chang, G. W. Harrington, P. D. Lotlikar, D. Hadjiolov, M. Levitt, S. Rajalakshmi, and D. S.R. Sarma, J . k t / . Cancer Inst.. 58. 263 (1977). S.K. Chang, G. W . Harrington, H. S. Veale, and D. Swern, J . Org. Chem., 41, 3752 (1976).
RECEIVED for review March 31, 1977. Resubmitted July 18, 1977. Accepted September 12, 1977. Presented a t the 11th MARM, April 1977, Newark, Del. T h e investigation was supported by P H S Research Grant CA-18618 from the National Cancer Institute.
Fourier Transform Infrared Analysis below the One-Nanogram Level R. Cournoyer," J. C. Shearer, and D. H. Anderson Industrial Laboratory, Eastman Kodak Company, Rochester, New York
The interfacing of microscopy with Fourier Transform infrared (FTIR) spectroscopy Is a useful combination allowing samples of less than 1 ng to be identified. The sample, usually supported by a thin sodium chloride plate, is centered in a aperture 50 to 200 pm in diameter. The sample mount is oriented in an 8X beam condenser In an FTIR spectrometer where multiscan signal averaging techniques produce a spectrun wlth the desired signal-to-noise ratio. One or two hours of total analysis time Is generally required. Polymers and other solids as well as oils and various liquids have been Identified. The small amounts of material require that the entire sample preparation be done under a microscope.
T h e characterization of samples too small to be visible to the naked eye is restricted to the domain of the microscope. In many cases characterizations can be made accurately and quickly with t h e microscope alone, b u t some microsamples d o not yield to such analysis or do so only with great difficulty. These samples require the interfacing of modern instrumental techniques with classical microscopic analysis. The interfacing of microscopy with Fourier Transform infrared ( F T I R ) spectroscopy is a useful combination allowing preparations of less t h a n 1 ng of sample to be identified. T h e sample, usually supported by a thin sodium chloride plate, is centered
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in an aperture 50 to 200 ,urn in diameter. T h e sample mount is oriented in an 8X beam condenser in an FTIR spectrometer where multiscan signal averaging techniques produce a spectrum with the desired signal-to-noise ratio. One or two hours of total analysis time is generally required. Polymers and other solids as well as oils and various liquids have been identified using this approach. EXPERIMENTAL Instrumentation. A stereo and compound microscope as well as microtomes, hot stages, or any other apparatus appropriate for the particular samples at hand are required. A fine pointed probe (dissecting needle), forceps, and other paraphernalia suitable for micromanipulation are also necessary. A Digilab FTS-14 Fourier Transform spectrometer equipped with the standard nichrome wire source and TGS detector, and a Perkin-Elmer 8X reflecting beam condenser were used to produce the spectra. Sodium Chloride Plates. Thin sodium chloride plates (200-500 pm thick) are prepared by cleaving rock salt used in standard infrared work. The salt crystals are first cut into rectangles (approximately x cm) with a clean, single edged stainless steel blade. This rectangle is transferred with forceps to a clean microscope slide and placed under a stereo microscope. The rectangle is stood on edge and cleaved into two plates of equal thickness with a clean, unused single edged blade. The process is repeated until plates of the desired thickness are obtained. Plates without both faces freshly cleaved are discarded to avoid possible contamination. A plate is selected that is flat and has flawless domains, and is trimmed to 1-2 X 3-4 mm. Some infrared ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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salt plates will not cleave satisfactorily and much aggravation can be avoided by searching out plates that are easily cleaved. A single (38 X 19 X 4 mm) infrared rock salt crystal will provide scores of the desired thin plates. Cleanliness throughout the preparation is imperative because of the small amount of sample involved in the analysis. Aperture Disk. Perkin-Elmer micropellet disks with 250 or 500 pm apertures are the bases for sample mounts. Apertures, 50 to 200 pm, are fashioned under the microscope by punching holes of appropriate diameter in 1-mil brass or stainless steel shim stock with tapered watchmaker‘s reamers. The apertures should be flat and have clean edges. Some polishing is necessary with an Arkansas stone. The aperture, centered on the micropellet disk, is attached with glue or adhesive tape. The aperture disk surface and aperture should be inspected under the microscope for cleanliness prior to use. S a m p l e P r e p a r a t i o n . The technique used for sample preparation is determined by the nature of the sample, the available equipment, and the manipulative skills and preferences of the microscopist. The sample should be prepared so that it will approximately fill the aperture, and so that it is only several micrometers thick. The thin salt plates are used to support the sample in an aperture of appropriate diameter. The smaller samples require the use of the smaller apertures. The sample may be too thin or, as more often the case, too thick. A general approach has been to make the sample as thin as possible. One approach for solids has been to place the sample on a microscope slide and roll it under a fine pointed probe tip; another approach is to press the sample between two clean microscope slides or a slide and a coverslip, then use a new, solvent washed razor blade to peel the material from the glass surface. Another technique which is suitable for solids and liquids is to place the sample between two freshly cleaved thin salt plates and apply gentle pressure with a probe. The plates must be flat and without irregularities; otherwise the pressure applied will crack the plates. When the two salt plates are separated, most or all of the sample generally remains on one of the plates. If the sample will not stay flat because of elasticity or surface tension, the plates need not be separated. Troublesome samples sometimes require the use of a small vise to hold the salt plates together. Liquid sample handling is sometimes facilitated by transferring the liquid to a flexible film. The film is then arched with the liquid at the high point, and the sample transferred by touching the salt plate to the droplet. These are only the most generally applicable sample preparation techniques used in our laboratory. Other techniques exist and additional ones are continually being developed as different samples require. Small amounts of volatile materials may be handled by adapting cryogenic techniques familiar to many microscopists and other workers involved with low temperature sample manipulation. Sample Mounting. The sample side of the salt plate is placed against the aperture disk surface when a single salt plate is being used. With this orientation, the sample sits in the aperture rather than some distance above it. The thin salt plates with the samples attached are secured to the aperture disks with a small amount of soft wax. The wax is applied to the end of the salt plate surfaces with a fine pointed probe. An aperture disk containing the sample is then placed in the FTIR spectrometer, and aligned by adjusting the disk position until the detector signal is maximized. FTIR spectra are accumulated until a spectrum with usable signal to noise ratio results. Higher gains and longer scanning times are required as the sample or aperture size decreases. We have found 50 pm to be the smallest usable aperture for our instrument. A Globar source and/or Mercury-Cadmium-Telluride detector will enable the use of somewhat smaller apertures; however, diffraction effects will prevent the use of apertures less than 30 pm.
RESULTS AND DISCUSSION T h e sensitivity of t h e FTIR spectrometer has enabled infrared identification of increasingly smaller amounts of material (1-3). T h e exploitation of this sensitivity requires new approaches for sample presentation t o the instrument. Investigations carried o u t in this laboratory indicate t h a t miniaturization of t h e familiar K B r pellet would not give suitable preparations for samples at t h e nanogram level 2276
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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Figure 1. Spectrum of 6 ng of triphenyl phosphate in a 100-wm aperture scanned 1000 times at 8 cm-’ resolution
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Figure 2. Spectrum of 3.4 ng of cellulose acetate with 10% triphenyl phosphate in a 100-pm aperture scanned 2000 times at 8 cm-’ resolution
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Figure 3. Spectrum of 0.9 ng of cellulose acetate with 10% triphenyl phosphate in a 50-pm aperture scanned 2000 times at 8 cm-’ resolution
because of dilution and loss of sample, or impurities in t h e KBr or solvents. Even when these limitations were overcome, atmospheric contamination would often swamp out the sample signal. A single atmospheric “dust speck” is frequently 10
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Figure 4. Spectrum of 0.9 ng of cellulose acetate with 10% triphenyl phosphate in a 50-pm aperture scanned 96000 times at 8 cm-' resolution
to 100 times the weight of the samples being considered. It was decided for these reasons that the samples would have to be examined neat. Sodium chloride was chosen as the most suitable support for the sample because of its infrared transmission a n d ease of cleavage. T h e use of microscopic techniques throughout the sample preparation has two advantages; the analyst is able to work with materials not visible to t h e naked eye, a n d microscopic physical and chemical separations enable relatively pure samples to be used, which greatly increases the probability of successful identification by infrared spectroscopy. Figure 1 is an infrared curve of 6 ng of triphenyl phosphate. This curve was used to identify an isolated 6-ng sample found in a manufacturing environment. Total analysis time was approximately 2 h of which 80% involved sample preparation. Figure 2 is the spectrum of 3.4 ng of cellulose acetate film base in a 100-pm aperture, a n d that in Figure 3 is of 0.9 ng
of the same sample in a 50-pm aperture. Both of these spectra are unsmoothed and were recorded after 2000 scans. T h e increased noise in Figure 3 shows a reduction in sample size and aperture. T h e spectrum in Figure 3 is identifiable as cellulose acetate even though less t h a n 1 h of spectrometer time and less t h a n 1 ng of sample were used. Figure 4 shows the improvement obtained by prolonged scanning (-40 h) of the sample described in Figure 3. T h e noise has been reduced to the extent that absorptions due to 90 pg of triphenyl phosphate, which is present a t approximately 10% in the film base, are recognizable. These absorptions have been marked with x's and may be compared with those in Figure 1. Microscopic sample manipulation combined with t h e sensitivity of F T I R spectroscopy allows us to identify previously unidentifiably small amounts of material. Work is under way to construct a stronger beam condenser to reduce d a t a collection time and to improve sensitivity. T h e combination of this condenser with refinements in sample preparation should result in a t least a n additional 10-fold reduction in minimum identifiable sample size. T h e obtainable spectra are of sufficiently high quality that spectral subtractions will be feasible. Work is now under way to demonstrate the practicality and utility of this technique. Successful infrared spectroscopic analysis of materials at the nanogram to picogram level indicates that this technique, with all its advantages, can be considered as an ultra micro method.
ACKNOWLEDGMENT We thank Rex Wooton for his assistance in producing the spectra in Figures 1 through 4.
LITERATURE CITED (1) P. R. Griffiths and F. Block, Paper 329, 23rd Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, 1972. ( 2 ) S. S. T. King, J . Agric. Food Chern., 21, 526 (1973). (3) D. H. Anderson and T. E. Wilson, Ana,. Chem., 47, 2482 (1975).
RECEIVED for review July 18, 1977. Accepted September 26, 1977.
Spectrophotometric Determination of Acrylonitrile Maynard E. Hall" and John W. Stevens, Jr.' Univeristy of Arkansas, Graduate Institute of Technology, Post Office Box 30 17, Little Rock, Arkansas
A spectrophotometric method for the determination of acrylonitrile based on the absorbance of visible light of a pyridine-acrylonitrile complex at 41 1 nm has been developed. This complex Is formed in the presence of a basic hypochlorite solution at 60-65 OC. The molar absorptivity, based on acrylonitrile concentration, is 635.4. The precision of the method is f3.18% of the acrylonltrile present in the 5-30 ppm range. The complex color is stable for at least 20 mln after development.
T h e usefulness underlying t h e chemical analysis of acrylonitrile lies in the fact that it is widely used in the 'Present address, Dow Chemical Company, P.O. Box 520, Magnolia, Ark. 7 2 7 5 3 .
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synthetic rubber, fiber, and plastic industries. In the United States alone in 1976 over 1.5 billion pounds were produced ( I ) . Sensitive and reliable analytical methods for acrylonitrile are necessary for economic and toxicological purposes. Acrylonitrile is known to be toxic when ingested, inhaled, or applied to the skin, and the U.S. Public Health Service has set the maximum allowable limit for the monomer in the air at 20 p p m (2). Recent studies by Du P o n t have linked acrylonitrile to cancer in workers and steps are being taken by Du P o n t to reduce exposure to workers to 2 ppm ( 3 ) . Analytical procedures which have been used most for acrylonitrile are titrimetric (4-6), gas chromatographic (7-9), and polarographic (10-13). T h e titrimetric procedures are good for relatively high concentrations while the others can measure as little as 5 ppm in aqueous solutions. T h e few gas chromatographic procedures reported are sensitive b u t have been limited to nonaqueous systems. Polarographic methods ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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