Anal. Chem. 1990, 62, 2247-2251
of dynamic spectral data is particularly interesting in its potential application to exploratory, nondestructive analysis of fluorescent mixtures, since no assumptions regarding spectral shape or lifetime are necessary. We are currently exploring the application of frequency-domain fluorescence techniques to the analysis of more complex samples.
ACKNOWLEDGMENT We appreciate the participation of Donald S. Burdick and Xin M. Tu of the Institute of Statistics and Decision Sciences and Department of Mathematics a t Duke University in the data analysis.
LITERATURE CITED (1) Warner, I. M. In Comtempcvary Topics in Ana/ytitxl a d c/jnjm/ Chemistry: Hercules, D. M., Hieftje, G. M.. Snyder, L. R., Evenson, M.
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A.. Eds.; Plenum Press: New York, 1982; Vol. 4. (2) Russell, M. D.;Gouterman, M. Spectrochim. Acta 1988, 44A. 857. (3) Russell, M. D.;Gouterman. M. Spectrochm. Acta 1988, 44A. 863. (4) Russell, M. D.: Gouterman, M.; Van Zee,J. A. Spectrochh. Acta 1988, 44A. 873. (5) Millican, D. W.; McGown, L. B. Anal. Chem. 1989, 67. 580. D.S.; Tu, X. M.: McGown, L. 6.: Millican. D. W.J . Chemom. Burdick, (6) 1990, 4 , 15. (7) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York. 1983. (8) Mattheis. J. R.; Mitchell, G. W.; Spencer, R. D. In New Direcffons in Mdeculer Luminescence; Eastwood, D., Ed.; ASTM: Philadelphia, PA, 1983. (9) McGown, L. B.; Millican, D. W. Appl. Spectrosc. 1988. 42, 1084. (10) Burdick, D. S.; Tu, X. M. J . Chemom. 1989, 3 , 431.
RECEIVEDfor review May 11, 1990. Accepted July 19, 1990. This work Wa5 supported by the United States Department of Energy (Grant NO. DE-FG05-88ER13931).
Transient Infrared Transmission Spectroscopy Roger W. Jones* and John F. McClelland Center for Advanced Technology Development, Iowa State University, Ames, Iowa 50011
Transient Infrared transmlsslon spectroscopy is a new methad that can acquke analytkally useful transmigslon spectra from movlng, optlcally thlck sollds. No sample Preparation Is required. The spectra are of sufflclent quality for accurate quantitatlve composnional analysis. The method works by the creatlon of a thin, short-lived, chilled layer at the sample surface. Blackbody-ilke thermal emisskn from the bulk of the sample Is selecthfely absorbed as it passes through the chilled layer, so the transmlsslon spectrum of the layer Is superimposed on the observed thermal emission. Spectra of polycarbonate, beeswax, and copolymers of methyl and butyl methacrylate are presented. Composltlonal analysis of the methacrylate copolymers with a standard error of predlctlon of only 0.87 mol % Is demonstrated.
INTRODUCTION The analysis of solids by infrared spectroscopy has long been problematic because of the high optical density of most solids a t infrared wavelengths, but methods have been developed to cope with the difficulties. On the other hand, if the analysis must also be done in real time on moving sample material, such as on an industrial process line, it may not be possible by any method. Conventional transmission spectroscopy requires dilution or physical thinning to lower the optical density of the sample, but this precludes real-time analysis, is labor intensive, and destroys sample integrity. Diffuse reflectance can examine moving solids in real time without sample preparation but is largely limited to powders and places limits on the optical scattering, optical absorption, and morphological properties of the sample material (I, 2). Photoacoustic spectroscopy can be applied to solids with a wide range of morphologies and without sample preparation, but the sample material must be acoustically isolated, limiting it to near real-time analysis and precluding on-line applications
* To whom correspondence should be addressed.
(1,s).Conventional emission spectroscopy is limited by optical density the same way transmission spectroscopy is. High optical density induces self-absorption and obscures the spectrum, so conventional emission spectroscopy is limited to optically thin samples (4). Recently a new method called transient infrared emission spectroscopy (TIRES), which can analyze in real time optically thick solids in motion, has been under development (5-7). In TIRES an optically thin surface layer of the sample is rapidly heated for an instant and thermal emission from this layer is collected before it thickens and cools by thermal diffusion. This reduces self-absorption to levels that allow quantitative spectra and results to be obtained. Although TIRES works well on a wide variety of materials, it does rely on surface heating and on sensing thermal emission. Accordingly, the hotter the surface is, or more precisely, the greater the thermal gradient between the surface and the bulk is, the better. This means TIRES may not be applicable to certain thermally sensitive materials or to streams of hot material, such as often occur in industrial process settings. Transient infrared spectroscopy in general, however, only requires a sharp, near-surface thermal gradient to produce an optically thin surface layer not in thermal equilibrium with the sample bulk. The surface may be either hotter or colder than the bulk. We introduce in this paper a technique called transient infrared transmission spectroscopy (TIRTS) which uses a chilled surface layer. In TIRTS, a jet of cold gas or some other cold source rapidly cools the surface of a moving stream of solid sample material within the field of view of a spectrometer. This produces an optically thin, chilled layer a t the sample surface within the field of view. The layer thickens and warms by thermal diffusion, but as it does so it is also carried out of the field of view by the sample motion. As a result, the chilled layer within the spectrometer field of view remains thin. The uncooled, optically thick bulk of the sample acts as the infrared source of TIRTS since it behaves as a blackbody infraredemission source. The thermal emission from the bulk must pass through the chilled surface layer to reach the spectrom-
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Laboratory apparatus for TIRTS. A jet of liquid-nkogencooled helium Strikes the rotating sample disk wnhin the field of view of the spectrometer. A jet of heated nnrogen retums the sample suface to its wiglnal temperatue. A heat gun warms h a back of me sample disk for experiments wnh elevated sample temperatures. Flgure 1.
eter. Like all objects above 0 K, the surface layer both emits and absorbs radiation, hut since it is colder than the bulk, its net effect is the partial absorption of the infrared light passing through it. Since the layer is optically thin, the absorption is selective. Like a properly prepared sample placed in the beam of an infrared spectrophotometer, the surface layer impresses its structured transmission spectrum on the light passing through it. In TIRTS, the infrared emission reaching the spectrometer consists of a blackbody envelope characteristic of the temperature of the sample hulk, with the transmission spectrum of the surface layer superimposed on it. Many experimental parameters affect TIRTS the same way that they do TIRES. For example, the amount of saturation (caused by self-absorption) in TIRES spectra is largely controlled by how long the emission is observed after the surface layer is heated 6 6 ) . TIRTS has a similar dependence because the chilled surface layer starts to thicken immediately after being formed. Accordingly, the longer the chilled layer is observed by the spectrometer after its forming, the greater the average observed thickness of the layer will he, and the higher the observed optical density of the layer will he. The faster the sample material is moved through the spectrometer field of view, either by reducing the size of the field of view or by increasing the sample speed, the leas saturation results. TIRTS shares with TIRES many of the same strengths. Both are single-ended, real-time techniques that can work on moving samples without sample preparation. Both me largely insensitive to sample morphology, to optical properties other than the desired absorption coefficients (e.g., scattering coefficients, reflectivity), and to sample environment (e.g., sample hacking, surrounding atmosphere).
EXPERIMENTAL SECTION The experimental arrangement used for TIRTS is very similar to that for TIRES with a hot jet (7)except that the positions of the hot and cold jets are interchanged. Figure 1shows the setup
for TIRTS. The normal infrared source of a Perkin-Elmer 1800 spectrophotometerwas removed and a disk of the sample material was put in its place so that the spectrophotometer viewed the sample normal to its surface. The sample disk was mounted on the shaft of a variable-speed motor and spun to simulate a continuous flow of fresh material through the spectrometer field of view. A KCI window covered the spectrometer port, hut no other optics were used. The spectrometer had a wide-hand liquidnitrogen-cooled HgCdTe detector (D*= 1 X 1O'O em Hz'12 W-I), operated at a 1.50cm/s optical-path-differencevelocity and Bm-' nominal resolution, and accumulated 256 scans for each spwtrum. Helium was chilled by passage through a liquid-nitrogen bath at 0.10 to 0.14 L/s. The stream of cold helium was directed onto the sample surface within the spectrometer field of view by a 1 mm inner diameter tube. The end of the tube was within 2 mm of the sample surface and was positioned a t a 4 5 O angle with respect to both the sample surface and the direction of motion of the sample through the field of view. Downstream from the field of view a jet of heated nitrogen was directed onto the chilled surface track left by the cold jet. The temperature and flow rate of the nitrogen were adjusted so that the nitrogen raised the temperature of the sample surface hack to near i t s value prior to passage through the cold jet. In this way the rotating sample could mimic a continuous flow of uniform-temperature material into the spectrometer field of view and cold jet. During experiments on samples above room temperature, a heat gun was directed onto the hack of the sample disk to raise the hulk sample temperature. The heat gun was removed for tests of room-temperature samples. Conventional transmission spectra must he converted into transmittance spectra by ratioing against a blank in order to compensate for the response function of the spectrometer and the emission curve of the infrared source. The same is true of TIRTS transmission spectra. For TIRTS spectra, the hlackbody-like emission curves ohserved from the rotating samples before the cold and hot jets are tumed on act as the blanks. These emission curves for polycarbonate and beeswax are shown in Figures 2 and 5. A defect in our spectrometer produces a small voltage spike in the interferograms. The spike is so small that it is imperceptible in the interferogramsthemselves, but it shows itself as a very low amplitude sine wave oscillation on all of the Fourier-transformed spectra. The oscillations are invisible in Figures 2 and 5 because they are so small, and they tend to cancel out when two spectra are ratioed. They cannot be completely ignored, however, because they produce zero and negative values in the weak, high-wavenumber tails of the blank emission and TIRTS spectra. To avoid division by zero and negative numbers when converting to transmittance,very small constants were added to both the blank and the TIRTS spectra before ratioing, as was done previously for some TIRES spectra (5). In each case the constant was equal to 0.2% of the spectrum maximum or less. Some of the raw transmittance spectra had sloping baselines. which have been corrected. A flat baseline would result only if the effective sample temperatures for the blank emission curve and the TIRTS spectrum were the same. This can occur, but in our lab experiments it was difficult to achieve both because of the range of chilled-layer temperatures and thicknesses within the spectrometer field of view and because it would require a precise balancing of the cold snd hot jets. The helium cold jet displaced water vapor and carbon dioxide from the optical path; thus the TIRTS transmission spectra were largely free of contamination from these sources. The blank emission spectra were generally not as clean, so some of the resulting raw transmittance spectra had positive-going water and carbon dioxide features. These have been removed by spectral subtraction from some of the transmittance spectra in Figures 3 and 4. In addition, nine-point (18 cm-') SavitzkyColay smoothing was used to make the spectra in Figures 3 through 5 clearer at higher wavenumbers. It should he noted that addition of small mnstants, c a r h a dioxide spectral subtraction, and smoothing affect only the data above Zoo0 cm-'. These manipulations are not needed if only the fingerprint region is of interest The blank emission and TIRTS transmission spectra (Figure 2 and the bottom of Figure 5 ) are presented on the same vertical scale except as explicitly marked. This is not true for the transmittance spectra. Because the thickness and temperature
ANALYTICAL CHEMISTRY, VOL. 62, NO. 20, OCTOBER 15, 1990
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gradient of the chilled surface layer varies with experimental conditions (e.g., sample speed, sample temperature) and also varies within the field of view of the spectrometer at any given moment, the absolute transmittance values observed are not related to sample composition. Only the relative transmittances within each spectrum are meaningful. Accordingly, no ordinate scale is shown in Figures 3 through 6. The transmittance spectra in Figures 3 through 5 have been vertically offset and scaled to appear the same size to aid in comparing them. The spectra in Figure 6 have not been scaled, only offset. As a result, the bottom and top of each of these figures do not correspond to 0 and 100% transmittance, respectively, for any of the spectra. Others have observed that it is necessary to take into account emission from the spectrometer itself when measuring the emittance spectra of relatively low-temperature samples (8,9). A similar consideration should formally apply to TIRTS spectra. The spectrometer emission becomes less important, however, as the intensity difference between the emission sample and the blackbody standard decreases (8). In our TIRTS experiments, the overall intensities of the TLRTS and blank spectra do not differ by a large amount. In addition, we have found that the contribution made by our spectrometer is largely equivalent to a room-temperature graybody 180" out of phase from the sample signal. As a result, including the effect of the spectrometer has little effect on the relative transmittances observed, and we have chosen not to compensate for the contribution from spectrometer emission in our spectra. Quantitative analysis was done with commercial principalcomponent-regression (PCR) software (CIRCOM from PerkinElmer (10-12)). The data manipulations described in the previous paragraph are largely cosmetic and are not necessary for PCR analysis. Prior to PCR analysis, the transmission spectra were scaled to a constant total intensity and then were converted to transmittance-like spectra by ratioing them against a room-temperature blackbody spectrum from carbon black. The carbon black spectrum was used for all to avoid introducing variations from ratioing each TIRTS spectrum against its own blank emission spectrum. Use of carbon black also demonstrates the unimportance of the material used for the blank. The blank spectrum need not be changed as the sample composition changes. No constants were added to the spectra before ratioing and no other data pretreatment was done. The samples of poly[(methyl methacrylate)-co-(butyl methacrylate)] used for the quantitative experiments are the same set described in more detail previously (7). The compositions of the disks were known to *0.4 mol %. Reference transmittance spectra are included in Figures 3 and 5 for comparison. These spectra were not recorded by conventional transmission because the samples were optically thick (and for beeswax, highly scattering) at infrared wavelengths. Instead, absorbance spectra were recorded with an MTEC Model 200 photoacoustic detector mounted in the spectrophotometer (with its normal infrared source in place), and the absorbance spectra were converted mathematically to transmittance. The absorbance
Flgure 3. TIRTS transmittance spectra of 3-mm-thick polycarbonate traveling at 40.8 cm/s derived by ratioing the TIRTS transmission and emission spectra in Figure 2, and a reference transmittance spectrum recorded photoacoustically.
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spectra were recorded at 0.05 cm/s optical-path-differencevelocity and 8-cm-' nominal resolution by accumulating 32 scans.
RESULTS AND DISCUSSION Figure 2 shows both emission and TIRTS spectra for 3mm-thick polycarbonate (Lexan) traveling a t 40.8 cm/s through the spectrometer field of view. The spectra have not been corrected for the response function of the spectrometer and detector. The two emission curves were recorded with the sample at a uniform temperature, so they are very blackbody-like. The 23 "C emission spectrum is virtually identical with that of a blackbody, while the 132 "C spectrum has some small structure characteristic of polycarbonate (most prominently around 1200 cm-'). When the cold jet is turned on, however, the observed emission changes drastically as the transmission spectrum of the thin, cold layer is superimposed on the emission, giving the TIRTS transmission spectra shown. The TIRTS transmission spectra have all of the characteristic features of polycarbonate, although the relative sizes of the features are greatly affected by the variation in intensity of the blackbody emission. The emission and TIRTS transmission spectra in Figure 2 were ratioed to produce the transmittance spectra in Figure 3. The noise level in the TIRTS spectra increases with increasing wavenumber because they are the ratio of spectra that decrease in intensity with increasing wavenumber. In addition, the signal-to-noise ratio is lower in the 23 "C spectrum because the intensities of the original spectra in Figure 2 were lower. Despite all this, the C-H stretch bands around 3000 cm-' are visible in both TIRTS spectra and all features below 3000 cm-' in the reference spectrum are also present in the 132 "C TIRTS spectrum. The spectra in Figure 3, both TIRTS and photoacoustic, suffer from obvious saturation. As described in the Introduction, the saturation in TIRTS spectra can be reduced by increasing sample speed or reducing the spectrometer field of view so that the average thickness of the chilled layer within the field of view is reduced. Figure 4 shows TIRTS spectra of the same polycarbonate disk at the same bulk temperatures, but at a 408 cm/s sample velocity. Obviously, the spectra in Figure 4 display less saturation than those in Figure 3, including the reference photoacoustic spectrum. At the higher sample speed the chilled layer is both thinner and not as cold, so the strength of the absorptions is reduced. Accordingly,
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WAVE NUMBER S Flgure 6. TIRTS transmittance spectra of 40.8 cm/s, 3-mm-thick poly[(methyl methacrylatekco-(butyI methacrylate)] samples with a 23 "C bulk temperature. Compositions are (top to bottom) 100.0, 93.1, 85.7, 77.8, 69.2, 60.0, 50.0, 39.1, 27.3, 14.3,and 0.0mol % methyl methacrylate.
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Figure 5. Spectra of 40.8 cm/s, 3-mm-thick beeswax made with a uniform sample temperature (emission), with a chilled surface layer (TIRTS transmission), and by ratioing the emission and TIRTS transmission spectra (TIRTS transmittance). A transmittance spectrum recorded photoacoustically is included for reference.
the higher-speed spectra have smaller signal-to-noise ratios. Since TIRTS works by reducing the surface temperature, it can be applied to heat-sensitive materials. As an example of that, Figure 5 shows spectra for yellow beeswax (Fisher Scientific) which melts a t 62 to 65 "C (13)and which is optically a highly scattering material. The beeswax was a disk 3 mm thick on average, at room temperature, and moving at 40.8 cm/s through the spectrometer field of view. The TIRTS transmittance spectrum shown was derived by ratioing the emission and TIRTS transmission spectra in the figure. Within the fingerprint region the TIRTS spectrum compares well with the reference photoacoustic spectrum. To test the quantitative ability of TIRTS, the spectra of 11 copolymers of methyl methacrylate and n-butyl methacrylate were recorded by TIRTS and analyzed by principal component regression (PCR). The sample disks averaged 3 mm thick and had the compositions 0.0, 14.3, 27.3, 39.1, 50.0, 60.0, 69.2, 77.8, 85.7, 93.1, and 100.0 mol % methyl methacrylate. The TIRTS spectra were recorded with the disks at room temperature (23 "C) and moving at 40.8 cm/s. The
Flgure 7. Compositions of copolymers of methyl and butyl methacrylate predicted by cross validating principal component regressions of the 1100 to 790 cm-' region of the spectra in Figure 6 plotted against the known sample compositions. The standard error of prediction is 0.87 mol%.
transmittance spectra are arranged in Figure 6 in order of composition with pure poly(methy1 methacrylate) a t the top and pure poly(buty1methacrylate) at the bottom. The spectra have obvious composition-dependent features, but there is extensive overlap of the methyl-related and butyl-related bands. A PCR cross validation was carried out on only the 1100-790 cm-l range of the spectra (14, 15). In the cross validation, each of the samples was chosen in turn as the unknown, the other ten samples acted as standards to calibrate the regression, and then the regression was used to predict the composition of the unknown. The 11 predicted compositions are plotted in Figure 7 against the true compositions known from the synthesis of the samples. The standard error of prediction, which is the root-mean-square deviation of the predicted values from the true values (14, 15),is only 0.87 mol %.
The results presented here show that TIRTS produces quantitatively accurate spectra from optically thick, moving solids. Quantitative analysis is possible even a t the low thermal-emission intensities from room temperature samples. The addition of TIRTS broadens the range of materials accessible to transient infrared spectroscopy. TIRTS and TIRES are two manifestations of the general concept of
Anal. Chem. 1990, 62, 22151-2256
transient infrared spectroscopy in which a dynamic thermal gradient is induced so that the spectroscopic behavior of an optically thin layer of material differs from that of the rest of the sample. Like TIRES, TIRTS overcomes the problem of high optical density in solids that previously prevented the real-time infrared analysis of most solid samples. TIRTS and TIRES are insensitive to the reflectance and optical-scattering properties of samples, and function in real time without sample preparation. Unlike TIRES, TIRTS does not involve raising sample temperature, so it can be applied where elevated temperatures cannot be used.
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(7) Jones, Roger W.; McClelland. John F. Anal. Chem. 1990, 62, 2074-2079. (8) Kember, D.;Chenery, D. H.; Sheppard, N.; Fell, J. Spectrochlm. Acta, Part A 1979. 35, 455-459. (9) Brown. R. J.; Young, 8. G. Appl. Opt. 1975, 14, 2927-2934. Fredericks, Peter M.: Osborn, Paul R.; Swlnkels, Dom A. J. Anal. (10) Chem. 1985, 57, 1947-1950. (11) Frederlcks, Peter M.; Moxon, Neville T. Fuel 1986, 65, 1531-1538. (12) Malinowski, Edmund R.; Howery, Darryl G. Factor Anawsls In Chemist ~Wiley: ; New York, 1980. (13) The Merck Index, 10th ed.;Wlndholz, Martha, Ed.; Merck: Rahway, NJ, 1983. (14) Haaland, David M.; Thomas, Edward V. Anal. Chem. 1988, 6 0 , 1193-1202. (15) Haaland, David M.; Thomas, Edward V. Anal. Chem. 1888, 6 0 , 1202- 1208.
LITERATURE CITED Grifflths, Peter R.; Fuller, Michael P. I n Advances in InfraredandRamen spectroscopy: Clark, R. J. H., Hester, R. E., Eds.; Heyden: London, 1982; Vol. 9, pp 63-129. Fraser, David J. J.; Griffihs, Peter R. Appl. Spectrosc. 1990, 4 4 , 193-199. McClelland, John F. Anal. Chem. 1983, 55, 89A-105A. Grifflths. Peter R. Appl. Spectrosc. 1972, 26, 73-76. Jones, Roger W.; McClelland, John F. Anal. Chem. 1989, 6 1 , 650-656. Jones, Roger W.; McClelland, John F. Anal. Chem. 1989, 6 1 , 1810-18 15.
RECEIVED for review May 21,1990. Accepted July 27,1990. This work was funded by the Center for Advanced Technology Development, which is operated for the U.S. Department of Commerce by Iowa State University under Grant No. ITA 87-02 and in part (J.F.M.) by Ames Laboratory, which is operated for the U.S. Department of Energy by Iowa State University under Contract No. W-7405-ENG-82, supported by the Assistant Secretary for Fossil Energy.
Integrated Optical Attenuated Total Reflection Spectrometry of Aqueous Superstrates Using Prism-Coupled Polymer Waveguides S. S. Saavedra and W. M. hichert* Department of Biomedical Engineering and Center f o r Emerging Cardiovascular Technologies, Duke University, Durham, North Carolina 27706
Attenuated total reflectlon (ATR) spectrometry of aqueous solutions In contact wlth polystyrene integrated optlcai waveguides has been Investigated. The mode-dependent absorption of evanescent energy by fluorescein solutions adjacent to the waveguide sutface was measured and compared to theoretical predlctlons based on a ray optlcs approach. Although enhanced sensitlvlty was observed with increaslng mode number, the sensttivlty for the highest order mode was less than that predicted by theory.
INTRODUCTION Attenuated total reflection (ATR) spectrometry is a wellestablished technique for obtaining absorbance spectra of opaque samples and thin films and for examining interfacial phenomena (I). The major limitation of ATR spectrometry is that its sensitivity is typically 3-4 orders of magnitude less than conventional transmission measurements using a 1-cm path length cell. This lower sensitivity is due to the small penetration depth of the evanescent wave into the absorbing medium, typically around 100 nm in the UV and visible regions of the spectrum. One approach to enhancing the sensitivity of ATR spectrometry is to increase interaction of the evanescent wave with the absorbing medium by increasing the number of reflections per unit distance along the internal reflection element (IRE)/sample interface. Since the number of reflections per unit distance is inversely proportional to IRE thickness, substantial enhancements in sensitivity are observed for very 0003-2700/90/0362-2251$02.50/0
thin IRES. This mode of enhancement was recently employed by Stephens and Bohn (2),who measured the absorption of evanescent energy by monolayers bound to the surface of a 150 pm thick glass coverslip. Even greater sensitivity can be realized with integrated optical (IO) waveguides that have thicknesses comparable to the wavelength of the propagating light and therefore support hundreds to thousands of internal reflections per centimeter (3, 4 ) . IO waveguides, particularly those fabricated from thin, transparent polymer films of micron dimensions, have attracted considerable interest as a means of acquiring Raman spectra from monolayers deposited on the waveguide surface or of the waveguide material itself (5-9). In spite of the low efficiency of Raman scattering, IO waveguide Raman spectroscopy is a feasible experiment due to (1)the extremely high optical field intensities that can be generated in the guide and (2) the increased path length created by the large number of internal reflections per centimeter. The concept of integrated optical waveguide ATR (IOWATR) spectrometry was discussed from a theoretical perspective by Midwinter almost 20 years ago (3). However, the only experimental application of IOW-ATR spectrometry to date was reported by Mitchell (41, who showed that aqueous solutions of bilirubin delivered to the surface of an IO waveguide through a flow cell attenuated the guided mode intensity via absorption of the evanescent wave. A major limitation of this approach was the significant refractive index discontinuities created by placing the in- and outcoupling prisms outside of the flow cell volume. These discontinuities essentially limited efficient prism to prism propagation to the 0 1990 American Chemical Society
