2427
Anal. Chem. 1988. 60. 2427-2435
then removed by using spectral subtraction. The absorbance of the carbonyl peak in the final spectra is perfectly linear. This indicates that the spectral coaddition method can be reliably used for quantitative measurements. The spectra features necessary for identification of methyl palmitate are visible a t the 10-ng level. With this criterion for an identification limit, this is an MIL of 10 ng. Application. The SFC/FT-IR flow cell interface described here has been used in a variety of applications including the analysis of thermally labile carbamate pesticides (19-21) and natural products (20,22). In each application, FT-IR identification of the eluted components shows high sensitivity for these compounds and provides positive identification to distinguish between related species.
LITERATURE CITED Jinno, K. Chromatograph& 1987, 2 3 , 55-62. Shafer, K. H.; Pentoney, S. L.; Grifflths, P. R. HRC CC, J . High Reso/ut. Chromatogr . Chromatogr . Commun. 1984, 7 , 707-709. Fujimoto. C.; Hirata. Y. H.; Jinno, K. J. Chromatogr. 1985. 332, 47-56. Shafer, K. H.; Pentoney, S. L.; Griffiths. P. R. Anal. Chem. 1988, 58, 58-64. Shafer. K. H.; Pentoney, S. L.; Griffiths, P. R.; Fuoco, R. HRC CC, J . High Resolut . Chromatogr . Chromatogr . Commun . 1988. 9 , 168-171. Pentoney, S. L.; Shafer, K. H.; Griffiths, P. R. J. Chromatogr. Sci. 1988, 2 4 , 230-235.
Raynor, M. W.; Bartie, K. D.; Davies, I. L.; Williams, A.; Clifford, A. A,; Chalmers, J. M.; Cook, B. W. Anal. Chem. 1988, 60, 427-433. Shafer, K. H.; Griffiths, P. R. Anal. Chem. 1983, 55, 1939-1942. Oiesik, S. V.; French, S. B.; Novotny, M. Chmmatcgraphie 1984, 18, 489-495. Johnson, C. C.; Jordan, J. W.; Taylor, L. T.; Vidrine, D. W. Chromatograph& 1985, 20, 717-723. Hughes, M. E.; Fasching. J. L. J. Chromatogr. Sci. 1985, 2 4 , 535-540. French, S. B.; Novotny, M. Anal. Chem. 1988, 58, 164-166. Peaden. P. A.; Lee, M. L. J. Chromatogr. 1983, 259, 1-16. Fiekls, S. M.; Lee, M. L. J. Chromtogr. 1985, 349, 305. Morin, P.; Caude, M.; Richard, H.; Rosset, R. Chromatograph& 1988, 2 1 , 523-530. Levy, J. M.; Rltchey, W. M. HRC C C , J . High Resolut. Chromatogr. Chromatogr. Commun . 1987, IO, 493-496. Smith, R. D.; Kaiinoski. H. T.; Udseth, H. R.; Wright, B. W. Anal. Chem. 1984, 56, 2476-2480. Snyder, L. R.; Kirkland, J. J. Introduction to Modern UquH Chromatography, 2nd ed.; Wiley: New York, 1979; pp 31-33. Wieboidt. R. C. Ana&& of PestlcHes by Cap//&ry SFC-FTIR; Nicolet FTIR Application Note AN-8705, 1967. Wieboldt, R. C.; Smlth, J. A. I n Supercrltical FluH €xtraction and Chromatography-Techniques and Appiicabns ; Charpentler, B. A., Sevenants. M. R., Eds.; ACS Symposium Series 3 6 6 Amerlcan Chem ical Soclefy: Washington, DC, 1988; pp 229-242. Later, D.W.; Bornhop. D. J.; Lee, E. D.; Henion, J. D.; Wieboldt, R. C. LC-GC 1987. 5 . 804-816. Wleboldt, R. C.; Kempfert, K. D.; Later, D. W.; Campbell, E. R., submitted for publication in HRC CC, J. High Resolut. Chromatogr. Chro-
.
matogr Common.
RECEIVED for review March 25,1988. Accepted August 2,1988.
Quantitative Effects of an Absorbing Matrix on Near-Infrared Diffuse Reflectance Spectra Jill M. Olinger and Peter R. Griffiths* Department of Chemistry, University of California]Riverside, California 92521
The absorptlon propertles of a matrix surrounding an analyte influence the band intenSny In near-lnfrared dmuSe reflectance spectra. I f the matrlx does not absorb radiation at the same wavelength as the analytical band, then use of the KubelkaMunk equation provldes a linear relationship between band lntenslty and concentration over a major portlon of the concentration range for the analyte. If the m a t h surrounding the analyte absorbs radlatlon at the same wavelength as the analytical band, then deviations from llnearlty of plots of the Kubeika-Munk function versus concentration occur. I n thls case, the use of log l/R’values instead of the Kubelka-Munk function has been shown emplrlcally to provide a more linear relatlonshlp between reflectance and concentration. I t has also been shown that the concentration range over which llnearlty holds Is dependent upon partlcle sire and on the strength of the absorptlon by the matrix. The reason for thls behavior Is explained by the effectlve penetratbn depth of the beam, whlch Is shown to be only one or two partlcle dlameters when absorption by the matrlx Is strong.
Quantitative analyses performed with absorption spectrometry usually depend upon a linear relationship between band intensity and concentration. For diffuse reflectance (DR) spectrometry, Kubelka-Munk (K-M) theory indicates that linear plots of band intensity versus concentration should result when intensities are plotted as the K-M function 0003-2700/88/0360-2427$01.50/0
F(R) = (1- R)2/(2R) where R is the absolute diffuse reflectance of the analyte at infinite depth. F(R) is the ratio of the absorption coefficient, K, to the scattering coefficient, S. S = 2s, where s is the scattering coefficient per centimeter in the absence of absorption. K is equal to twice the Beel-Lambert law absorption coefficient, lz. For a nonscattering neat sample of path length b cm, having a transmittance of T
k = In (1/7‘)/b
(2)
Therefore, according to K-M theory for dilute samples in a scattering matrix
(3) where a is the absorptivity and c is the concentration of the analyte. Use of the Kubelka-Munk equation for quantitative analysis by diffuse reflectance spectrometry is common for measurements made in the ultraviolet, visible (I, 2), and mid(3)and far-infrared (4) regions of the spectrum, but not in the near-infrared (near-IR) region. As has been pointed out in several review articles (5-7), since Norris made the earliest reports on using near-IR reflectance spectrometry for the quantitative determination of components in agricultural products (8,9), almost all near-infrared DR spectra have been converted to log 1/R values prior to utilization in a program 0 1988 American Chemical Society
2428
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
for multiple linear regression (MLR) analysis with the implication that Beer’s law, and not the K-M equation, is being followed. With the exception of one report by Geladi et al. (IO),little attention has been given in the literature to any parameter other than log 1/R for near-IR reflectance analysis involving the application of MLR techniques. The parameters that control near-IR diffuse reflectance spectra and for which the K-M theory is meant to provide a quantitative description are absorption, reflection, and scattering of radiation within a sample. One of the assumptions under which Beer’s law was derived is that reflection and scattering of radiation within the sample must be insignificant (11). It is therefore surprising that Kubelka-Munk values would not at least provide a more linear relationship with concentration than log 1/R. The assumptions in the derivation of the K-M equation, as given by Kortum ( I ) , are as follows: (1) the sample is illuminated with monochromatic radiation; (2) the distribution of scattered radiation is isotropic so that all regular (specular) reflection is ignored; (3) the particles in the sample layer are randomly distributed (i.e. the particles are distributed homogeneously throughout the sample); (4)the particles are very much smaller than the thickness of the sample layer itself; (5) the sample layer is subject only to diffuse irradiation; (6) the particles are much larger than the wavelength of irradiation (so that the scattering coefficient will be independent of wavelength), although if only one wavelength is observed this point is not relevant; (7) the breadth of the macroscopic sample surface is great compared to the physical depth of the sample or the diameter of the beam focus (to discriminate against edge effects). Most of these assumptions appear to be met in practice. Provided that the width of the instrument line-shape function (e.g. the slit function of a monochromator) is much narrower than the widths of the absorption bands, the assumption of monochromatic radiation is still valid. Discrimination against regular reflectance may be achieved by careful choice of the geometry of the optics (12,13). Although samples are never illuminated by diffuse radiation, it is commonly believed that as long as the radiation penetrates through several particle layers, the incident radiation is quickly diffused due to multiple scattering and reflection. A further assumption in the derivation of eq 1 is that the reflectance is measured for a sample that is of “infinite” depth, i.e. when a further increase in the depth of the sample causes no change in the measured reflectance. One of the primary factors not mentioned in the above list of assumptions that can cause deviation from eq 3 is that in practice, a relative reflectance, R’, rather than an absolute reflectance is usually measured. The relative reflectance is equal to the ratio zs/zR, where Is is the intensity of radiation reflected from the sample and ZR is the intensity of radiation reflected from a reference material. In the case of near-IR reflectance spectrometry this material is usually a ceramic disk. Strictly speaking, the K-M equation requires that an absolute reflectance, defined as the ratio Is/&, be measured, where Zs is defined as above and Zo is the intensity of diffuse incident radiation. The measurement of absolute reflectance can only be made directly through the use of an integrating sphere, and even then some care is required. An absolute reflectance can be derived from relative reflectances although the derivation requires several experimental measurements (1).
Kortiim (1, 14) has shown that the use of relative reflectances in the visible region can cause deviations from K-M theory if the analyte is surrounded by an absorbing matrix. This effect was shown by measuring the reflectance of Crz03 in an absorbing matrix relative both to the pure diluent and to a highly reflective standard (MgO). When the pure diluent
was used as the reference, a plot of F(R? vs concentration of Crz03was nonlinear. When MgO was used as the reference, a similar plot yielded a straight line with a positive intercept. Kortum states that a straight line with a zero intercept can only be obtained when F(RA) is plotted vs c, where
RAis the absolute reflectance of the analyte (A), R ~ + isMthe
+
reflectance of the analyte matrix (A+M) relative to the reflectance of the matrix (M), p is the absolute reflectance of a nonabsorbing standard, and R‘Mis the reflectance of the matrix relative to the reflectance of the nonabsorbing standard. For most near-IR reflectance analyses of cereal products, it is impossible to measure either R b or R ~ + due M to the complexity of the sample and the impossibility of obtaining a separate measurement of the matrix surrounding any analyte. It is generally accepted that the K-M equation, like Beer’s law, is a limiting equation and should only apply for weakly absorbing bands, i.e. when the product of absorptivity and concentration is low. For organic materials, absorptions in the near-infrared region are due to vibrational overtones and combination bands. The absorptivities of these bands are much weaker than the absorptivities of the corresponding fundamental vibrations. Thus most organic analytes can be considered to be weakly absorbing in the near-IR region even without dilution. As noted above, however, for most near-IR analyses, the analyte (such as protein or lipid molecules in a cereal) is usually not isolated from other components but is surrounded by a matrix that not only is complex but also often absorbs the incident radiation at least as strongly as the analyte at the analytical wavelengths. In a cereal product analysis, for example, the matrix would largely consist of carbohydrate molecules. It would therefore be expected that unless the proper referencing method is used, absorption by the matrix surrounding the analyte may cause deviations from eq 2. The effect of an absorbing matrix on the linearity of plots of Kubelka-Munk band intensities versus concentration was also studied by Brimmer and Griffiths for the mid-infrared region (15). They found that plots of F(R? versus concentration for an analyte dispersed in a nonabsorbing matrix were linear for analytes at concentrations below about 5 w t % provided that the absorptivity of the band was not excessively high. Even for samples in nonabsorbing matrices, these authors showed that R ’increased because of front surface reflection (Fresnel reflection) for bands of high absorptivity (12,13, 16). With optimized optical configurations for the mid-infrared studies, this concentration range could be extended to about 50% for all but the most intense bands (16). When the analyte is dispersed in an absorbing matrix, however, the plot of F(R? versus concentration assumes a sigmoidal shape for fundamental bands in the mid-infrared region (15). (It is possible that similar results would have been obtained in the visible region had Kortum (14) extended the concentration range of his study.) The sigmoidal shape of the plots in ref 15 suggests that even the use of log l/R’instead of F(R9 would not provide a linear plot when the matrix absorbs incident radiation at the same wavelength as the analyte. A correction such as suggested by eq 4 was not shown. In light of the differences between the quantitative behavior of diffuse reflectance spectra in the visible and mid-infrared regions, and because of the growing importance of near-IR reflectance spectrometry, the effect of absorbing matrices in this spectral region has been investigated and is reported below.
EXPERIMENTAL SECTION Up to three components, carbazole (Aldrich),NaCl (Fisher),
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
and carbon (ground graphite electrodes obtained from Bay Carbon, Inc.) were mixed during the preparation of each sample. Carbazole is intended to represent a typical organic analyte since it contains both C-H and N-H bonds. It was used without further purification or grinding and was sieved to particle size ranges of 18-53 and 75-125 pm by using a Sonic Sifter (ATM Corp.). NaCl and graphite represent nonabsorbing and absorbing components of the matrix, respectively. NaCl and the graphite electrodeswere each ground and sieved separately into the same size ranges as the analyte. The NaCl was dried in a 150 “C oven before use. Graphite was not dried, to preclude air oxidation. Each component was weighed separately and then mixed together in a glass vial by rolling and shaking the vial by hand. Samples were packed into 3 mm deep, 16 mm diameter stainless steel cups by pouring a predetermined volume into the cup and loosely packing the sample by applying slight pressure with a flat object. Care was taken not to tamp the sample as this would alter the packing density, thus changing the scattering coefficient ( I , 17). Samples used to model the behavior in a nonabsorbing matrix consisted of binary mixtures of carbazole and NaCl. Samples used to model the effects of an absorbing matrix consisted of carbazole dispersed in NaCl with either 1 or 5 wt % of graphite added. Reflectance spectra were calculated by ratioing the spectrum of the sample against a spectrum of the diluent that contained the same weight percent graphite. If the sample contained 2% carbazole, y% graphite, and 2% NaCl, the reference was prepared with ( x + z ) % NaCl and y% graphite. For samples consisting of carbazole in an absorbing matrix it was true that ( y / ( y + 2)) x 100 = 1 or 5 wt % as appropriate. In this way, the reference should have the same reflectance as the matrix surrounding the analyte. Samples prepared in this way are referred to as being ratioed against a weight-corrected reference. Since the carbazole particles have been replaced with an equal weight percent of NaC1, the reflectance of the reference should always be greater than or equal to that of the sample at all wavelengths. In practice, this behavior was not always observed when the single-beam spectrum of a sample in an absorbing matrix was ratioed against that of a weight-corrected reference. In all cases, however, the base line was closer to 100% reflectance then for spectra for which the reference was either pure NaCl or a standard 1or 5 w t % mixture of graphite in NaC1. Spectra were measured on a Pacific Scientific 6250 grating monochromator equipped with a fiber-optic accessory and PbS detector. The fiber-optic accessory provided a beam diameter of approximately 3 mm, greatly reducing the quantity of sample required for each measurement in comparison to the standard sample cup provided with this spectrometer while still allowing a representative sample to be measured. A total of 700 data points were collected at equal wavelength intervals between 1100 and 2500 nm. The fixed slits of this instrument provide a 10-nm nominal band-pass at 2500 nm, which increases slightly at shorter wavelengths. Each spectrum is the ensemble average of 100 scans. Corrections for base-line variations due to packing differences and possible particle size variations were performed by subtracting the value in a region of minimal absorption by the carbazole analyte (1238 nm) from the value corresponding to the band of interest (usually 1672 nm). When a sloping base line was encountered, an equivalent correction was performed after linear interpolation between two wavelengths at which absorption by the analyte was minimal. The use of second derivative spectrometry for base-line corrections was also studied. A segment size of 6 nm and gap size of 0 nm was used. Where plots of band intensity versus concentration are shown with error bars, the upper and lower bounds indicate the highest and lowest value, respectively, of three measurements(repacking$. The middle value is the average of the three measurements. Lines on the graphs are least-squaresbest fits of a polynomial (between second and fifth order) to the average values. The degree of the polynomial chosen to represent the best fit lines is not meant to have any physical meaning but was chosen only to show a smooth curve through the data. Transm’issionmeasurements to determine absorptivities were made on a Nicolet 740 FT-IR spectrometer equipped with a CaFz beamsplitter and InSb detector. The 13 mm diameter pellets were pressed at 40 OOO psi for 10% carbazole in NaCl and at 49 OOO psi for 0.82% graphite in KBr for approximately 90-120 s.
2429
0.1954 0.19
-
A
0.185. 0.18 0.1751
0.4
1
0.3
0.1
i
0. t i
1.1
1.3
1.5
1.7
1.9
2.1
2.3
2.5
WAVELENGTH ( ~ 1 Nu) 0 ~
Flgure 1. Absorbance spectra of (A) 1 wt % graphite dispersed in
NaCl and (B) neat carbazole.
RESULTS AND DISCUSSION Quantitative Behavior. The spectrum of neat carbazole (particle size 18-53 pm) is shown in Figure 1, together with an ordinate-expanded spectrum of the 1wt % graphite matrix (particle size 18-53 pm). It can be seen that absorption by the dispersion of graphite in sodium chloride is approximately, but not exactly, uniform across the region 1100-2500 nm. The increased noise at the longer wavelengths is due both to attenuation of the radiation by the optical fibers and to decreased response of the detector. The small apparent band near 1500 nm is an instrumental artifact caused by Wood’s anomaly. Some absorption due to moisture is seen near 1920 nm. Absorption by the added graphite increased markedly with particle size. For samples containing 1 wt % graphite, the average value of log l/R’was -0.17 for samples containing particles with diameters ranging between 18 and 53 pm and increased to -0.53 for 75-125 pm particles. A transmission measurement of 0.82% graphite in a KBr pellet showed the absorptivity of graphite to be approximately 2000 cm-I across most of the near-infrared spectrum. This absorptivity indicates that any photon entering a graphite particle of the diameter used in this study is completely absorbed and thus cannot be detected. The carbazole band a t 1672 nm (indicated by an arrow in Figure 1B) was chosen to study the linearity of plots of band intensity versus concentration since it was identifiable above the noise level even tor samples containing the lowest concentration of carbazole (1 wt %) in the most absorbing matrix (5 wt %). This band absorbs in a region where the base line was relatively flat for all samples. The absorptivity of carbazole at 1672 nm was measured as 15 cm-l; therefore even particles as large as 100 pm in diameter have a transmittance of 70%. After base-line correction as described in the Experimental Section, F(R? and log 11R’were plotted against concentration. The results for the particle diameters in the range from 18 to 53 pm are shown in parts A and B of Figure 2, respectively, for the three matrices studied, i.e. pure NaC1,l w t % graphite in NaCl, and 5 wt % graphite in NaC1. Although the data over the entire concentration range in both plots are better fit by a higher-order polynomial than a straight line, the Kubelka-Munk function provides the more linear plot for the
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
2430
O*l81 0.16
:::v ,
0
i5]
0.28
?zr
0.24
j
5 4
0.2
m
L I o u
2
E'
40 60 U T I CARBAZOLE
20
EO
0
0.16
0.08
0 -
0.4
0.6
0.8
1
Figure 3. (A) log 1lR' vs concentration; (B) F(R') vs concentration for graphite dispersed in NaCI. Particle size was 18-53 pm.
3 0.12
::
0.2
YSS GRAPHITE
t a 2 .
::I-
,
0
100
U
Y Y u 4
.
0.04 0 0
20
40 60 YTScMDUoLE
80
100
Figure 2. (A) F(R') vs concentration for carbazole dlspersed In (A) NaCI, (B)1 wt YO graphitelNaCI, and (C) 5 wt % graphitelNaClobtained by using the weightcorrectedreferencingmethod. Particle slze for all components of each sample was 18-53 pm. (8)log 1lR'and average number of carbazole particle diameters interrogated vs concentration for carbazole dlspersed in (A) NaCi, (B) 1 wt % graphitel NaCI, and (C) 5 wt % graphite/NaCI obtained by uslng the welghtcorrected referenclng method. Particle size for an componentsof each sample was 18-53 pm.
carbazole analyte in a nonabsorbing matrix. Conversely,log l/R'values provide the more linear plot over a major portion of the concentration range studied for carbazole in samples containing 1wt % graphite. For the samples containing 5 wt % graphite, neither F(R3 nor log 1/R' yield linear plots, although the plots of log l/R'versus concentrationof carbazole are more linear than the correspondingF(RJ' plots. It should be noted that, as expected, the use of second derivatives led to the same conclusions drawn from Figure 2. The variation of log l/R'and F(R9 with the concentration of graphite in NaCl up to 1w t % (in the absence of carbazole) is shown in lines A and B of Figure 3, respectively. Since plots of F(R3 vs concentration for carbazole in NaCl alone were linear, it might be expected that when only graphite is dispersed in NaCl, plots of F(R3 vs concentration would also be linear. As can be seen in Figure 3, however, the log 1/R' function certainly provides the more linear relationship. The behavior shown in Figures 2 and 3 is surprising in two respects. First, even though graphite has a high absorptivity, the concentrations are low enough that contributions to the signal from front surface reflection should be negligible, which should favor adherence to the K-M function. (At high concentration, the reflectance of graphite plateaus a t approximately 10% reflectance because of front surface reflection.) The second anomaly is that the absorption of radiation on addition of graphite to carbazole is not additive. If the absorption of radiation by graphite and carbazole were additive, the variation with concentration of F(R9 of backgroundcorrected analyte bands for carbazole surrounded by an absorbing matrix should be linear even though the concentration of graphite decreases as the concentration of carbazole increases.
Effect of Reference. As noted in the Experimental Section, all reflectance spectra were obtained by using a weight-corrected reference; i.e. the single-beam spectrum of the sample was ratioed against that of a matrix in which the percent graphite had been adjusted to match the actual weight percentage of graphite in the sample. In practice, however, most near-IR reflectance spectra are collected by simply ratioing the sample to a nonabsorbing reference, typically a ceramic disk. When the sample is prepared in an absorbing matrix, the base line of the spectrum will always occur at a value well below the reflectance of the ceramic disk. If the powdered analyte has been diluted in an absorbing matrix, the reference is often simply composed of the diluent. In this case, the base line of the ratio-recorded spectrum is always found at a reflectance greater than 100%. The results for carbazole in a 1 wt % graphite/NaCl matrix are similar to those of Kortum in the visible region obtained by using analogous referencing methods (14). Kortum, however, showed that if the spectra were obtained by ratioing against a highly reflective standard (MgO), linearity was obtained (albeit with a nonzero intercept). A study of the effects on near-IR band intensity caused by the choice of reference was therefore initiated. The effect of three types of reference was compared: (1) a weight-corrected reference; (2) the same graphite/NaCl mixture used to dilute the carbazole (i.e. 1 or 5 wt % graphite/NaCl); (3) a nonabsorbing material (NaCl). log 1/R' spectra of 8 wt % carbazole in 5 wt 7% graphite/NaCl referenced by all three methods are shown in Figure 4. Since the absorbance of graphite is approximately independent of wavelength, see Figure 1, base-line-corrected analyte band intensities in log l/R'spectra are approximately the same no matter which referencing method is used, even though large changes in the height of the base-line occur. For the spectrum calculated by ratioing against the spectrum of a weight-corrected reference (Figure4A) it can be seen that the reflectance of the reference does not match the reflectance of the matrix surrounding the carbazole since if this were true the base line would be at an ordinate value of zero. The magnitude of the shift in the base line away from zero showed no regularity with respect to a change in the concentration of carbazole. The reason for this irregularity is not understood, but might be caused by variations in the scattering coefficient because of inconsistent packing. The large negative shift in the base line for the spectrum calculated by ratioing against the spectrum of 5 wt % graphite/NaCl (Figure 433) is due to the reflectance of the sample being greater than the reflectance of the reference. For the spectrum calculated by ratioing against the spectrum of NaCl (Figure 4C), both a large positive shift and a slight downward curvature in the higher wavelength region of the base line are seen. Both of these effects can be attributed to the lack of
2431
ANALYTICAL CHEMISTRY, VOL. 80, NO. 21, NOVEMBER 1, 1988
A
0.287
1
-0.05
0
20
40 60 U T I CARBAZOLE
20
40
0.4-
80
100
B O.
0.41-
0.
0.4
.
0.
0.39
U
d
0.384
1.1
4
1.3
1.5
1.7
2.1
1.9
2.3
w 0.
2.5
-I 0
3
0.
YAVELENCTH ( X 10 WM) Flgure 4. Absorbance spectra of 8 wt % carbazole in a 5 wt % graphiWNaCI matrlx obtained by using as reference (A) a weightcorrected graphItelNaC1 mixture (the pure diiuent), (B)5 wt YO graphlte/NaCI, and (C) NaCI, respectlveiy. Particle size was 18-53 pm.
graphite in the reference to compensate for the graphite in the sample containing carbazole. The base-line problems caused by the choice of referencing method all point out the need to be able to do a correct base-line correction. If the Kubelka-Munk spectrum is calculated directly from any spectrum that has a base line above loo%, the bands will be distorted in the manner shown by Yeboah et al. (18). For weak bands, F(R3 can actually decrease as the analyte concentration increases. If, however, correction is done so that the base line in regions of minimal absorption from the analyte is always at R' = 1.00, then plots of either log 1/R' or F(R3 versus concentration will be approximately independent of the method of referencing. This is shown by comparing the plots of log l/R'for the 1672-nm band of carbazole versus concentration for carbazole dispersed in both the 1 and 5 wt 70 graphite/NaCl matrices with the three referencing methods studied (Figures 2B, 5A, and 5B). If plots of band intensity versus concentration were made for a band in the long wavelength region, where a base-line curvature occurs, extra care must be taken to compensate properly for the curvature and to measure the correct intensity in spectra referenced against NaC1. These results also show that since the range of linearity is approximately independent of the referencing method used, an incorrect choice of referencing method cannot account for the deviations from linearity of the type seen by Brimmer and Griffiths (15) in the mid-infrared region. Despite the fact that Kortum reported linear plots of F(R3 vs concentration when visible spectra of samples in an absorbing matrix were ratioed against the spectrum of a nonabsorbing material (MgO), the results obtained in this study for the same type of referencing method show that plots of log l/R'vs concentration are linear, while the corresponding plots of F(R3 vs. c are nonlinear. Although most samples for which near-IR reflectance analysis is used in practice are too complex for the direct application of eq 4, the system described in this paper is simple enough to derive absolute F(R) values. The values of R' needed are derived from the data shown in Figures 2A and 3B. The value for the absolute reflectance of NaCl at 1672 nm, p, was estimated as 0.96, using Figure 52 from ref 1. Varying the absolute reflectance by f4% led to little variation
0. 0.
0
60
80
100
UT% CARBAZOLE
Figure 5. (A) log l/R'vs concentration for carbazole in (A) 1 wt % graphite/NaCi and (B) 5 wt % graphIte/NaCI referenced against 1 wt % graphite In NaCl and 5 wt % graphite in NaCI, respecthrely. Particle size was 18-53 pm. (e) log l/R'vs concentration for carbazole in (A) 1 wt % graphite/NaCI and (B) 5 wt % graphite/NaCl referenced against NaCI. Particle size was 18-53 pm.
P, LL
& r
8
5 00
0
20
40
60
80
Ylt CARBAZOLE
Flgure 6. Absolute F(R) vs concentration as calculated per eq 4 for carbazole dispersed in (A) 1 wt % graphite/NaCi and (B) 5 wt % graphite/NaCI. Particle size was 18-53 pm.
in the final results. A plot of the absolute value of F(R) for carbazole derived by using eq 4 against concentration is shown in Figure 6. If the line corresponding to the absolute values of F(R) for carbazole in the 1 wt % graphite/NaCl matrix (Figure 6A) is compared to the plot for F(R 'j shown in Figure 2A, it is apparent that better linearity is obtained with F(R), especially at the lower concentrations. The correction technique described by eq 4 should yield plots that are independent of the graphite concentration. It can be seen, however, that the absolute values for carbazole in a 5 w t % graphite/NaCl matrix do not correspond exactly to those for carbazole in a 1w t % graphite/NaCl matrix. This result indicates that the "correction" to absolute values is not completely valid. The fact that F(R) for 90 w t % carbazole
2432
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988 A 0.4 1
I
0.25
t 0.2 LL
-
0
6
Oa4
20
40
60
100
80
1
0
2a
40
60
80
100
m hRBhZOLl Flgure 7. (A) F ( R ’ ) vs concentration for carbazole dispersed in (A) NaCI, (B) 1 wt % graphite/NaCI, and (C) 5 wt % graphRe/NaCI obtained by using the weightconected referencing method. Particle size for all components of each sample was 75-125 pm. (8)log l/R’and average number of carbazole particle demeters interrogated vs concentration for carbazole dispersed in (A) NaCI, (B) 1 wt % graphitel NaCI, and (C) 5 wt % graphWNaCl obtained by using the welghtcorrected referencing method. Particle size for all components of each sample was 75-125 pm.
in a 5 w t % graphite/NaCl matrix is less than F(R) for 70 w t % carbazole in the same matrix is probably due to a combination of irreproducibility of packing and a change in the slope of the plot of F(R? for graphite in NaCl vs concentration. Thus even for a simple system, the application of eq 4 to achieve a linear relationship between F(R)and concentration does not appear to be practical. Effect of Particle Size. Near-infraredreflectance analyses of cereal products are often performed by using particles with an average diameter of 100 Mm (6), whereas the results reported above are for somewhat smaller particles. In order to simulate the typical near-IR cereal analysis as much as possible, the same measurementswere made on samples for which the particles were in the size range 75-125 pm. The plots in parts A and B of Figure 7 are the equivalent plots to parts A and B of Figure 2 but for the larger particles. Two results are apparent: the noise level has increased and the linear range has decreased. The increase in the noise level can be explained by the fact that larger particles give rise to a greater Beer’s law absorption per particle. (The log l/R’values for a 1wt 70dispersion of 75-125-pm graphite particles in NaCl are almost double the corresponding values for the smaller particles.) Thus the increased absorption by the matrix containing larger particles decreases the signal at the detector by an amount that more than compensates for the increased absorption by the analyte. The signal-to-noise ratio (SNR) of the spectrum therefore decreases as the particle size increases. In addition, the use of the fiber-optic accessory means that somewhat less than 1000 particles are exposed to the incident radiation. Thus the sample is less likely to appear homogeneous or uniform
from one packing to the next even though the depth of penetration might have increased. The decrease in the range of linearity with the increase in particle size mertis a discussion on the probable interaction of the radiation with the sample in DR spectrometry. It is impossible to describe how each photon interacts with a diffusely reflecting powder due to the number of reflections, refractions, and diffractions that may occur in a sample containing irregularly shaped particles. Several theories have been developed to describe the overall effects observed in DR spectrometry ( I ) , of which the most usable one is the phenomenological theory of Kubelka and Munk (19). In the following discussion, Kubelka and Munk’s definition of scattering will be used, namely that scattered radiation is that component of the incident radiation that has been back reflected into the hemisphere whose flat boundary is parallel to the macroscopic sample plane. This is in contrast to the alternative definition given by Mie ( l ) i.e. , that any reflection or diffraction that occurs at the surface of a particle is scattering. Thus the scattering coefficient, s,in eq 3 is only related to radiation that is scattered in the sense defined by Kubelka and Munk. Despite the impossibility of describing how each photon interacts with a diffusely reflecting sample, it is possible to conceptualize certain events and their effects on the measured reflectance. When a weakly absorbing analyte (such as carbazole in the near-IR region) is completely surrounded by a nonabsorbing matrix (such as NaCl), the probability of radiation interacting with an analyte particle is great, since Fresnel reflection is generally low and the radiation should pass through the matrix, as shown schematically in Figure 8A. The reflectance of each NaCl particle can vary from about 4% for normal incidence to 100% at grazing incidence (20). After transmission through any particle (analyte or matrix) the radiation has a certain probability of being scattered back out of the sample to the detector with no further absorption by the analyte. The larger the particles, the greater is this probability. Since radiation incident on a nonabsorbing particle has a higher probability of being transmitted than being reflected according to the Fresnel equations, the probability of radiation being transmitted through several analyte particles is quite high especially if the particle diameter is small. The Kubelka-Munk equation was derived for such a sample (weakly absorbing analyte in a densely packed medium allowing multiple reflections and scatterings within the sample). As can be seen from Figures 2A and 7A, the K-M equation is followed quite well for carbazole dispersed in a nonabsorbing matrix. The effect of adding particles capable of strongly absorbing radiation at the same wavelength as the analyte can change this behavior. In this case, when radiation enters the sample and encounters one of the highly absorbing particles, the most probable event is that the radiation will be absorbed, and hence not detected (see Figure 8B). Radiation that has passed into the sample and has interacted with at least one analyte particle is less likely to be scattered out of the sample if it has the chance to be absorbed subsequently by one of the highly absorbing particles. Thus, as will be discussed quantitatively later, the average number of reflections occurring within the sample decreases. This decrease is apparently enough that the Kubelka-Munk theory, which assumes both a random distribution of particles within that part of the sample interrogated by the incident radiation and multiple scattering in the sample (I), should no longer provide a quantitative description of band intensities relative to concentration. The effects described above cannot be explained solely as changes occurring in the scattering coefficient unless the depth
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
2433
\
OETECTOR
A /
INCOMING RADIATION
I
EHERGIN6, SCATTERED RADIATION
0 . 3 4 . . 0
20
. 40- . 60. . 80. .
i
100
WTZ CARBAZOLE
Figure 9. Ratio of F(R') for 18-53-pm particles:75-125-pm particles for carbazole dispersed in (A) NaCI, (B) 1 wt (C) 5 wt % graphite/NaCI.
WON-ABSORBING PARTICLE
WEAKLY ABSORBING ANALYTE
Figure 8. (A) Schematic representation of possible reflections and transmissions a photon may undergo within a diffusely reflecting sample that contains only one weakly absorbing analyte surrounded by a nonabsorbing matrix. (B) schematic representation of possible reflections and transmissions a photon may undergo within a reflecting sample that contains one weakly absorbing analyte surrounded by an absorblng matrix.
of penetration has decreased to the point that radiation interrogates only one to two particle layers. Neither can it be ascribed to changes of the absorption coefficient for the analyte, although the overall absorption coefficient certainly decreases with decreasing graphite content. Instead the behavior is better explained by a change in the depth below the macroscopic plane of the sample at which the intensity of the incident beam has been reduced by a certain fraction. For isotropic samples, the distance into the sample at which the intensity of the incident beam, I,,, has been reduced to I o / e by absorption has been termed the optical absorption depth (21). An analogous nomenclature can be applied to powdered samples, even though scattering also tends to attenuate the beam. The penetration depth is inversely dependent on the concentrationof graphite. Thus the incident beam h a a much smaller probability of interacting with an analyte particle when the graphite concentration is high, and the intensity of the
YO graphite/NaCI, and
analyte bands in the DR spectrum is correspondingly weakened. Adherence to the behavior predicted by the K-M equation, as manifested by the linearity of plots of F(R3 vs concentration, is therefore likewise reduced. The effect of particle size on the optical absorption depth was studied by plotting the ratio of the K-M values for the two particle sizes examined vs concentration. (See Figure 9.) Curve 9A shows the results for carbazole in a nonabsorbing matrix. Except for the lowest concentrations, for which the fluctuations can be explained by the low SNR, the ratio remains essentially constant, indicating that the variation of the optical penetration depth with analyte concentration is independent of the particle size. These data suggest that for particles of a certain size the penetration depth of the beam is approximately constant at wavelengths that correspond to weakly absorbing bands in the near-IR region and is largely governed by scattering rather than absorption. The behavior in Figure 9A also suggests that the absorption coefficient remains constant regardless of the concentration of the carbazole. The fact that the ratio is less than unity is consistent with the generally accepted view that at wavelengths for which the absorptivity is low, the smaller the particle the lower will be the measured absorption. If the matrix is absorbing, the intensity ratio for both the 1and 5 wt 70graphite/NaCl matrices is greater than unity (up to 70 wt % graphite for the 1wt % graphite/NaCl matrix and up to 90 wt % for the 5 wt % graphite/NaCl matrix). The situation is further complicated by the fact that the absorption of the matrix itself changes from one analyte concentration to the next. Thus the extent to which the penetration depth changes from one sample to the next is not independent of particle size. This result is shown in Figure 9B,C and implies that the optical penetration depth will depend not only upon the proportion of absorber (graphite) to analyte (carbazole) but also on the particle size of the absorber. An estimate of the penetration depth can be made from the measured values of log (l/R'). Let us assume an average particle diameter, a, of 33 pm for the 18-53-pm particles and 100 pm for the 75-125-pm particles. Since the absorptivity of carbazole at 1672 nm is 15 cm-l, the absorbance of the 33 and 100 pm thick particles is 0.05 and 0.15, respectively. By dividing the base-line-corrected values of log 1/R' by the absorbance per particle, the effective number of carbazole particles through which the radiation reaching the detector has passed, N , can be calculated. This number is shown as the right-hand ordinate of Figures 2B and 7B, which show that even for the smaller particles, the beam does not interrogate the equivalent of more than six analyte crystals, and for the larger particles this number is reduced to about 2.3. For both the small and large particles, N is reduced even further if the matrix contains an absorbing component (graphite). The average number of carbazole and NaCl particle diameters through which those photons reaching the detector have
2434
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
h 1
\A
4 - . . . . . . . . ” 0
B
20
40 60 Yn CARBAZOLE
80
100
CONCLUSIONS
E ::i
o + - , 0
. . . .
20
10
, 60
yrs
CIUIUOLI
penetrates to the same depth for all analyte concentrations, the absorption should depend primarily on the number density of the analyte particles in the top layer and less on the scattering coefficient. In this case, log l/R’should be directly proportional to the analyte concentration provided that base-line correction is performed adequately. For samples prepared in a matrix of 5 w t % graphite/NaCl, N actually increases slightly when the carbazole concentration (as the graphite concentration decreases). In this case the effective penetration depth increases with carbazole concentration and plots of log l/R’vs c would be expected to be concave, and indeed this behavior is observed in practice; see Figures 2B.C and 7B.C.
. 80. .
,
100
Figure 10. (A) N v s concentration of carbazole for 18-53-pm particles dispersed in (A) NaCI, (8)1 wt % graphite/NaCi, and (C) 5 wt % of graphite/NaCl. (B) Corresponding plot for 75-125pm particles.
passed, N,can be estimated to a first approximation as 1OON/x,where x is defined in the Experimental Section. Plots of N vs carbazole concentration are shown in parts A and B of Figure 10 for the small and large particles, respectively. For samples where the analyte is present at low concentration in a nonabsorbing matrix, N is as large as 60 for the smaller particles (see Figure 10A.A) and 20 for the larger particles (see Figure 10B.A). The upper bound of the penetration depth is given by 0.5N& which in both cases is about 1 mm. Because of the effect of scattering, it is unlikely that the true penetration depth is much more than about one-third of this value, i.e. 300 pm. Thus for the smaller particles, the beam penetrates about 10 particle diameters below the macroscopic surface of the sample, while for the larger particles, the penetration depth is reduced to only three or four particle diameters. In the former case, this depth is apparently large enough that the assumptions made in the derivation of the Kubelka-Munk equation are fulfilled, so that a plot of F(R? vs c is linear for smaller particles in the absence of graphite; see Figure 2A.A. If the penetration depth is only three or four particle diameters, however, at least two of these assumptions, namely that the particles are very much smaller than the thickness of the sample layer and that the sample is subject to diffuse illumination, are of questionable validity. The increased curvature of the plot of F(R? vs c shown in Figure 7A.A in comparison to the plot shown in Figure 2A.A lends credence to this conclusion. For samples prepared with 1wt % graphite, N is surprisingly constant, having a value of approximately 10 for the 18-53-pm particles and 3 for the 75-125-pm particles; see Figures 10A.B and 10B.B, respectively. This result implies that the beam penetrates little more than one particle diameter into the sample, so that it is very unlikely that all the assumptions in the derivation of the Kubelka-Munk equation are fulfilled. This result also helps to explain why plots of log 1/R’ vs c are more linear than plots of F(R3 vs c , for samples prepared with 1 wt % graphite. Since the beam
Near-IR diffuse reflectance analysis is commonly applied to the determination of protein, oil, and moisture content in wheat and other cereal products by using log l/R‘values multiplied by coefficients determined by MLR to achieve quantitative results (5, 6). For example, for 75-125-pm particles including 1wt % graphite, the average value of log l/R’is 0.52. For wheat samples of slightly larger particle size, the value of log l/R’due to carbohydrate absorption a t the analytical wavelength for protein (2180 nm) is about 0.44 (22). The results of this study have indicated the reason why plots of log 1jR‘against analyte concentration are often more linear than the corresponding plot for F(R9. In many practical applications of near-IR reflectance spectrometry, the range of concentrations of the analytes, and hence the range of reflectance values, is sufficiently narrow that the effect of differences in the way that the ordinate is formatted is small. As the range of concentrations increases, the effect of the ordinate on the analytical result would be expected to become more significant. Some multiple regression routines allow the generation of calibration equations that compensate for residual nonlinearity by selecting another wavelength where the data have an equivalent nonlinearity. Nevertheless, as both the range of concentrations of the analyte and the number of analytes in each sample increase, it becomes more necessary to use a data format in which the ordinate varies linearly with concentration. Thus a good understanding of the factors relating band intensity to concentration is important in obtaining accurate analytical results for the widest variety of sample types.
ACKNOWLEDGMENT We gratefully acknowledge the donation of the Model 6250 spectrometer by Pacific Scientific.
LITERATURE CITED Kortum, Gustav Reflecrance Spectroscopy ; Springer-Verlag: New York, 1969. Wendlandt, W. W.; Hecht, H. G. Refbcfance Spectroscopy, Interscience: New York, 1966. Griffiths, Peter R.; Fuller, Michael P. A&. Infrared Raman Spectrosc. 1982, 9 ,63-129. Ferraro, John R.; Basil, Louis J. Fourier Transform Infrared Spectrosc o p y , Academic: Orlando, FL. 1985; Vol. 4, Chapter 6. Watson, C. A. Anal. C b e m . 1977, 49, 835A-840A. Wetzel, David L. Ana/. C b e m . 1983. 55, 1165A-1176A. Weyer, Louise G. Appl. Spectrosc. R e v . 1985. 21, 1-43. Massie. D. R.; Norrls, Karl H. Trans. ASAE 1965, 8 , 598. Ben-Gera, I.; Norris, Karl H. Isr. J. Agric. R e s . 1968. 18, 125-132. Geladi. P.; MacDougall, D.; Martens, H. Appl. Spectrosc. 1985, 39, 491-500. Willard, Hobart H.; Merritt, Lynne L.; Dean, John A.; Settle, Frank A. Instrumental Metbods of Analysis, 6th ed.; D. Van Nostrand: New York 1981; Chapter 3. Brimmer, Paul J.; Griffiths, Peter R.; Harrlck, N. James Appl. S p e c trosc. 1986. 40, 258-265, Brimmer, Paul J.; Grlffiths, Peter R. App/. Spectrosc. 1987, 4 7 , 791-797. Kortam, Gustav Nahvwiss8nscbaften 1966. 47, 600-609. Brimmer, Paul J.; Grifflths, Peter R. Anal. C h e m . 1988, 5 8 , 2179-2184. Brimmer, Paul J.; Grifflths, Peter R. Appl. Spectrosc. 1988, 4 2 , 242-247.
Anal. Chem. 1988, 60, 2435-2439 (17) Yeboah, Samuel A. Ph.D. dissertation, Ohio University, Athens, OH, 1982. (18) Yeboah. Samuel A,; Wang, Shih-Hsien; Griffihs, Peter R. Appl. SpectrOSC. 1084, 38, 259-264. (19) Kubelka, Paul: Munk, F. Z . Tech. Pbys. 1031, 72, 593-601. (20) Harrick, N. James Internal Reflection SpectroscopY: Wiley: 1967: p 5Q
(21) Rosenscwaig, A.; Gersho, A. J . Appl. Phys. 1078, 49, 2313.
2435
(22) Hruschka, W. R.; Norris, Karl, J. Appl. Spectrosc. 1062, 36, 26 1-265.
R ~ E I V Efor D review March 31,1988. Accepted August 2,1988. The work described in this paper was supported by CooperA~~~~~~~~ N ~ 58-43~y-6-0048 . from the u . ~De. partment of Agriculture.
Photochromism-Induced Photoacoustic Spectrometry for the Determination of Trace Mercury(II)as Its Dithizonate in the Solid State Nailin Chen, Runde Guo, and Edward P. C. Lai*
Department of Chemistry, Carleton University, Ottawa-Carleton Institute for Research and Graduate Studies in Chemistry, Ottawa, Ontario, Canada K1S 5B6
A new technlque, photochromlsm-Induced photoacoustlc spectrometry, has been developed for the determlnatlon of mercury( I I ) as mercury( I I ) dlthlzonate In the solld state. A speclal photoacoustic cell was designed for Inducing a photochromic reaction In mercury( I I ) dlthlzonate and detectlng the resultant photoacoustic signal from Its exclted state. Interferences from all other metal Ions are absent because only mercury( II ) dlthlzonate can exhiblt photochromlsm In the solid state. A linear standard calibration graph for Hg( I I ) was obtained In the quantlty range frgm 1.5 to 450 pmol. The relative standard devlatlons determined at 15 and 150 pmol were 6 % and 3%, respectlvely. When the technique Is applied to water analysls with dlthlzone extractlon, a detection limit of 3 pptr (parts per trllllon) Hg( I I)Is easily achievable.
As a toxic element, mercury is regularly determined in food, human blood, and environmental samples such as air, water, and soil. Various methods for the determination of mercury in water have been reviewed ( I ) . They include spectrophotometry, atomic absorption spectrometry, inductively coupled plasma (ICP) atomic emission spectrometry, fluorescence spectrometry, anodic stripping voltammetry, gas chromatography, and neutron activation analysis. In these methods, however, interferences are often encountered. Thus, separation or specific treatments are usually required before determination. In the study to be reported here, a new technique that is free from all metal ion interferences is described for the determination of Hg(11). I t is well-known that Hg(I1) is quantitatively transferred from an acidic aqueous solution to the organic phase as mercury(I1) dithizonate, H ~ ( H D z )by ~ , extraction with dithizone (2). Under the irradiation of strong visible light, Hg(HDt& undergoes a reversible photochromic reaction that is accompanied by a shift in its maximum absorption wavelength from 485 to 620 nm (3). The latter wavelength corresponds to an excited state of H ~ ( H D z )which ~ , can now be detected by photoacoustic spectrometry (PAS) using a He-Ne laser (output wavelength equals 632.8 nm). This technique
* Author to whom all correspondence should be addressed. 0003-2700/88/0380-2435$01.50/0
is called photochromism-induced photoacoustic spectrometry (PCPAS) of Hg(HDz)p PCPAS is best applied to solid H ~ ( H D zfor ) ~two reasons. First, since no photochromism is exhibited by any other metal dithizonates in the solid state (21, their presence will cause no interference with the analysis for Hg(I1). Second, our earlier work has shown that the microphone PAS sensitivity for solid samples is 2-3 orders of magnitude higher than that for solutions (4). Therefore, PCPAS of solid Hg(HDz), offers both advantages of specificity and high sensitivity. With this technique, the determination of 1.5 pmol of Hg(I1) is readily demonstrated.
EXPERIMENTAL SECTION Reagents. ACS grade reagents and deionized water were used. The extraction solution of 0.001-0.02% (w/v) dithizone in CC14 was stored, under cover with 0.5 M H2S04,for over 12 h before use. Since photochromism of solid H ~ ( H D zis) affected ~ by the characteristic quality of the solvent, certified ACS grade carbon tetrachloride (Fisher Scientific(2-187) was selected for this work. The other solvents studied were reagent ACS grade methylene chloride (AnachemiaAC-3172), certified reagent grade benzene (Caledon Laboratories 1600-1),and ACS spectro grade chloroform (Caledon). Sample Preparation. An aqueous solution of Hg(I1) in 0.5 M sulfuric acid was extracted with dithizone in CCb as described previously (5). Any intended interfering ions such as Ag(I), Au(III), Cu(II), and/or Pd(I1) were added to the solution before extraction. Excess dithizone in the extract was removed by washing with aqueous ammonia (1:lOOO dilution from Anachemia AC-622 reagent grade NH,OH, which contained 0.5 ppm heavy metals such as Pb). To prepare a solid film sample, a certain volume of the extract (e.g., 10 pL) containing Hg(HD& was delivered, drop by drop from a syringe, onto the center area of a microscopic cover glass of 18-mm diameter. At the same time, a gentle air stream was passed over the cover glass to speed up the drying of the extract. The resultant solid film of Hg(HDz), was generally less than 3 mm across, a size that could be easily covered by the incident He-Ne laser beam of 5-mm diameter. For the routine analysis of water samples, 0.5 mL of extract could be delivered by a computer-controlledsyringepump to form the solid film sample automatically (6). The solid film and its glass plate substrate were transferred to a PCPAS cell for analysis. Photoacoustic Measurements. The PAS setup was similar to that reported elsewhere (3). It consisted of a H e N e laser (18 mW), mechanical chopper, PCPAS cell, condenser microphone, 0 1988 American Chemical Society
