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Anal. Chem. 1981, 53, 2037-2040
Quantitative Photaacoustic Spectroscopy of PropranoloVMagnesium Carbonate Powder Mixtures in the Ultraviolet and the Near-Infrared Regions J.
K. Becconsall, Jonathan Percy, and R. F. Reld"
Imperial Chemical Industries Ltd., Corporate Laboratoty, P.O. Box 11, Runcorn, Cheshlre WA7 40E' Great Britain
To Investigate quantltatlve analysis of powder samples by photoacoustic spectroscopy, we measured the ultraviolet and near-infrared response of the heart drug propranolol at dlfferent concentrations in powder mixtures with magnesium carbonate. In the ultravloiet reglon, the response as a functlon of propranolol mass fraction Is markedly nonlinear. Results with mixtures prepared by cogrinding show that coatlng of propranolol onto magnesium carbonate particles contributes significantly to nonlinearity. Unground mlxtures stlll give nonllnear plots, whlch are dlscussed In terms of optlcai and thermal Interactions between the components. These interactions are considerably reduced In the near-infrared reglon, resulting In a linear dependence on propranolol mass fraction. When photoacoustic spectroscopy Is applied to quantltatlve analysis of formulated solld products such as pharmaceutlcals and pesticides, these effects must be taken into consideration.
In photoacoustic spectroscopy (PAS), the heat generated in a material following absorption of radiant energy is measured by the pressure change produced in an enclosed air space in contact with a sensitive microphone (1, 2). The great attraction of the technique is that spxtroscopic studies of solid samples are possible with little or no sample preparation, and a considerable number of qualitative measurements of the optical absorption characteristics of solids have been made with PAS (1-8). One situation where this could be of great importance is in the quality control laboratory. Quality control analysis of solids is, in general, very time-consuming, often requiring considerable sample purification and concentration prior to assay. A routine method for direct nondestructive assay of solid samples could be of great value. To meet this requirement, PAS must be shown to be suitable for quantitative as well as qualitative measurements. We report here our quantitative investigations of the photoacoustic response of powder mixtures of l-isopropylamin0-3-(l-naphthyloxy)propan-2-01(propranolol) with magnesium carbonate. Propranolol (structure shown in Figure 1) has activity as a P adrenergic blocker and is used in the Inderal tablet range produced by IC1 Pharmaceuticals Division (9). The example chosen for our investigation illustrates the potential of PAS for quality control analysis of formulated products, .such as pharmaceuticals and pesticides. EXPERIMENTAL SECTION The layout of the photoacoustic spectrometer is shown in Figure 2. The output from a 250-WXenon lamp (Applied Photophysics Ltd., Model 408/01)is passed through a f/4 grating monochromator (Applied Photophysics Ltd., Model M300),modulated by an optical chopper (Bentham Instruments Ltd., Model 278),and directed onto the sample tray of the photoacoustic cell (EDT Ltd., Model OAS 401). The output from the microphone is amplified (Bentham Model 213) and fed to a lock-in amplifier (Bentham Model 223), which provides synchronous detection of the photoacoustic signal. 0003-2700/81/0353-2037$01.25/0
To choose a suitable measurement wavelength, we fist recorded the UV-visible photoacoustic spectra of propranolol and magnesium carbonate. Using these spectra without any correction for the spectral power distribution of the lamp, monochromator, and cell, we found a peak signal at 318 nm in the propranolol spectrum, with a much smaller signal from magnesium carbonate at this wavelength. Test samples were made by mixing propranolol powder and magnesium carbonate powder together, either with or without grinding. The PAS signal intensity at 318 nm was then measured for each sample. Variations in lamp output intensity during the course of any given series of experiments were monitored and corrected for by repeatedly measuring the signal intensity at 318 nm from a carbon black reference sample during the experimental run. In the near-infrared region, complete spectra from 1.3 to 2.6 pm were recorded for all samples and corrected by calculating the ratio of the signal at each wavelength to that measured for a reference sample of carbon black. Propranolol showed PAS peaks due to the aromatic CH combination band at 2.2 pm and the aromatic CH overtone band at 1.72 pm. Magnesium carbonate gave peaks at 1.9 and 2.3 pm, The intensity readings used to characterize the propranolol content of the mixtures were made at 1.72 and at 2.2 pm. In each case the intensities were corrected for the contribution at these wavelengths from the magnesium carbonate spectrum.
RESULTS AND DISCUSSION Figure 3 shows a plot of the photoacoustic response at 318 nm of a series of propranolo1/MgCO3 mixtures containing between 0% and 100% propranolol. The powders in this caw were mixed and then coground. Clearly, for these samples, the photoacoustic intensity does not follow a linear relationship with mass fraction. It is likely that one important cause of this nonlinear behavior is the effect of cogrinding the two materials, whereby the softer propranolol becomes coated onto the harder magnesium carbonate particles. Particles thus coated produce an additional photoacoustic signal characteristic of propranolol, giving the appearance of an anomalously high propranolol content in the mixture. Cogrinding of a strongly absorbing powder with a nonabsorbing powder is already well-known in PAS as a means of alleviating saturation effects (10, 11). TOinvestigate the contribution which cogrinding makes to the curvature shown in Figure 3, we prepared a series of mixtures in which randomization of the powder particles was obtained by shaking rather than cogrinding. The photoacoustic response of these mixtures is illustrated in Figure 4. The dependence of the photoacoustic signal on the percentage of propranolol is still nonlinear, but the slope of the curve at low percentages is less than for coground samples. This is readily seen from the broken curve in Figure 4,which shows the response of the coground mixtures normalized to the intensity of the unground mixture at 100% propranolol. The coground mixtures show a stronger propranolol signal than the unground mixtures throughout the 0% to 60% range, confirming that cogrinding makes a significant contribution to the nonlinearity of the calibration curve, 0 1981 Amerlcan Chemical Society
2038
ANALYTICAL CHEMISTRY, VOL. 53, NO. 13, NOVEMBER 1981
Flgure 1. Structure of the
fl adrenergic blocker propranolol. P A. signal
MONOCHROMATOR
4 0-
/
SOURCE
/
OPTiCAL CHOPPER
CELL
I-..' 5 REFERENCE
i O/o
LOCK-IN
CHART
-A M P L I F I E R
AMPLIFIER
40
0
RECORDER
L
propranolol
80 (a/.)
Flgure 4. Photoacoustic signal at 318 nm for unground propranolol/ MgC03 mixtures compared with that for ground mixtures: (0)ground ground mixtures normalized to mlxtures; (A)unground mixtures; (0) match the unground mixtures response at 100% propranolol: (- -) dependence predicted by noninteractlng model. 8
Flgure 2. Schematic of the photoacoustic spectrometer.
0
PA.
e A.
signal
signal
2
40 0
40
80 '10
%propranolol (d-)
Figure 3. Photoacoustic signal at 318 nm as a function of % propranolol In ground propranolol/MgC03 mixtures.
In Figure 4 it is seen that the unground mixtures still do not show a linear dependence of the photoacoustic signal on propranolol content. This remaining nonlinearity must be attributed to the effects of the particulate nature of the sample on its radiation absorption and thermal relaxation behavior. In a bulk powder sample these effects are complicated by the depth of the sample. We have therefore made measurements 6n powder mixtures prepared as approximately single particle layers on adhesive tape. Figure 5 shows the response of a series of single particle layer samples of propranolo1/MgCO3mixtures, compared with the results from the bulk unground mixture. Evidently a considerable nonlinearity persists in the single layer sample. It is also interesting that throughout the concentration range the signal intensities from the single layer samples are only slightly less than those from the bulk powders, the reduction being at no point more than 20%. This suggests that in the bulk samples most of the photoacoustic signal is generated in the first particle layer, although this result must be interpreted cautiously since the monolayer sample differs in several respects from the top layer of a bulk sample. In particular, the signal intensity will be influenced
80
propranolol (+)
Figure 5. Photoacoustic slgnal at 318 nm for slngle particle layer samples compared with that for bulk unground samples: (0)slngle particle layers; (A)bulk samples; ( - e -) dependence predicted by noninteracting model.
by the different optical and thermal effects of the two backing materials (tape in one case and more of the same powder mixture in the other case). We must now examine the causes of the nonlinear behavior shown in Figures 4 and 5. We consider a binary random mixture of particles in which one component absorbs relatively strongly, and the other component absorbs weakly, a t the wavelength of the incident radiation. The simplest theoretical model, though one far removed from reality for most powder mixtures, is that in which the particles are assumed not to interact, either optically or thermally. For this simple model we expect the photoacoustic response to be proportional to the sum of the products: (exposed cross sectional area) X (photoacousticresponse per unit area) for the two componenb, which is a linear function of the exposed area of the strongly absorbing component and, therefore, of its bulk volume fraction in the mixture. It should be noted that the volume fraction is different from the mass fraction except at the 0% and 100% limits, if the two components have appreciably different bulk powder
ANALYTICAL CHEMISTRY, VOL. 53, NO. 13, NOVEMBER 1981
densities. In the case of the mixtures used in our study, we measured the bulk powder densities of propranolol and magnesium carbonate as 0.70 and 0.72 g cmT3,respectively. For these mixtures, therefore, the densities are so nearly the same that the volume fraction is virtually identical with the mass fraction, with the result that the above simple noninteracting imodel gives a linear dependence on mass fraction, as shown by the dot-dash line in Figures 4 and 5. For a mixture of components with different bulk powder densities, the prediction from this noninteracting model would be a curve. For the propranolol/MgC03 mixtures, the observed behavior (Figures 4 and 5) departs considerably from the linear response predicted by the simple model. The curvature observed can be attributed to optical and thermal interactions between the propranolol and the magnesium carbonate. Considering first the optical effects, a particulate mixture will absorb, scatter, and, depending on the absorption coefficients, tr,ansmit the incident light. The two most important processes in the system described are absorption and scattering. Absorbed energy produces a photoacoustic signal which depends OM the efficiency of the nonradiative deactivation of the absorbing molecule and on the thermal characteristics of the sample. If direct absorption were the only source of photoacoustic signal (noninteracting model), then the photoacoustic response would vary linearly with the volume fraction of' the strongly absorbing component. However, in a mixture, the weakly absorbing component, in this case magnesium carbonate, produces considerable scattering of the incident light. Some of this scattered light is absorbed by the strongly absorbing material (propranolol), giving rise to a larger photoacoustic signal than would be predicted by the noninteracting model. The additional photoacoustic signal produced as a result of light scattering will diminish as the fraction of the more strongly scattering component (magnesium carbonate) is reduced. Thermal interactions are also expected to contribute to nonlinearity, because the more strongly absorbing propranolol particles will be a t a higher temperature than the weakly absorbing magnesium carbonate particles. Thus in samples with high magnesium carbonate content, each propranolol particle is surrounded by a large heat sink of cooler gas and particles, allowing multidirectional heat flow out from the propranolol particle, with a resultant increase in the photoacoustic signal. At high propranolol concentrations such temperature gradients do not arise, and the heat flow is essentially undirectional. Both the light scattering and thermal diffusion effects can be considered in terms of the overall energy flow (radiation and heat) into and out from a propranolol particle. For a 100% propranolol sample there is, by symmetry, no net flow of either radiation or heat between each particle and its identical neighbors in the same particle layer (disregarding effects near the edge of the illuminated sample area). On the other hand, in a powder mixture containing only a low percentage of propranolol, each propranolol particle is surrounded by many mlagnesium carbonate particles which generate an additional lateral flow of scattered radiation into the propranolol particle, and at the same time allow an additional lateral heat flow out of the particle. Both these effects lead to an enhanced photoacoustic response. These mechanisms of enhancement through interaction between propranolol and magnesium carbonate obviously require the presence of both componenb, and the additional signal generated is therefore zero at the 0% propranolol and 100% propranolol points, reaching a maximum a t some intermediate concentration. A comparison of the gradients of the curves in Figure 4 at near 0% propranolol gives some measure of the signal en-
2039
hancement produced by cogrinding, light scattering, and thermal effects. The gradient for ground mixtures (broken curve, normalized) approaches a value at 0% propranolol of about 14 times that given by the noninteracting model (dot-dash line), while the corresponding gradient for unground mixtures (the lower of the two solid curves) is about 5 times that for the noninteracting model. We expect the effects to combine together in a multiplicative way in this region, Le., the 14 times enhancement factor should be the product of the factors for the three effects. We thus estimate that in the region near 0% propranolol we are obtaining enhancement factors of the following: for light scattering and thermal diffusion effects together, about 5 times, and for the cogrinding effect alone, about 1415 = 2.8 times. No adequate theoretical treatment exists to account quantitatively for the enhancement effects described above. Published treatments of the photoacoustic effect are based on the Rosencwaig-Gersho theory (12) and assume a planar sample surface with energy flows confined to the direction perpendicular to this plane. Formidable problems are encountered in trying to extend this model to particulate samples. Furthermore, experimental separation of thermal effects and light scattering effects seems virtually impossible. Nonlinear concentration dependence in PAS should not be confused with the photoacoustic saturation effects to which various authors have drawn attention (11-13). There is no direct connection between saturation and the nonlinear concentration dependences which we report here. In our measurements we obtained well-resolved spectral features under all conditions, including 100% propranolol, showing that photoacoustic saturation did not occur in this case. Furthermore, Freeman et al. (13)found in their experiments with powdered K2Cr207,which shows saturation, that simple mixing with a diluent powder without grinding did not remove thLe saturation effects. Therefore there is no reason to expect that, even where saturation exists, it will influence the dependence of signal on concentration in unground mixtures. Quantitative measurements by PAS on unground powder mixtures are determined solely by the number of absorbing particles and their environment, regardless of whether or not each particle is in a saturation condition. Additional evidence that thermal diffusion and light scattering are the principal nonlinear effects in the unground powder mixtures comes from the near-infrared photoacoustic response of the mixtures. Light scattering processes which are very marked in the UV photoacoustic response will be considerably reduced in the near-infrared spectra, since the efficiency of the scattering processes diminishes with increasing wavelength. Furthermore the absorption coefficients of propranolol and MgC03 are similar in the near-infrared region, resulting in smaller temperature gradients between particles in this region, and thus less interparticle thermal diffusion. Figure 6a shows the change of intensity of the aromatic CH combination band at 2.2 pm with propranolol content of a series of unground mixtures, whilst Figure 6b shows the same relationship for the aromatic overtone band at 1.72 pm. Both plots show a linear dependence of the photoacoustic response on propranolol content. This illustrates that the trends seen for the W responses are not caused by inconsistencies in smple mixing but are intimately related to the nature of the IJV photoacoustic response of powder mixtures. Further experimental and theoretical work to elucidate the mechanisms involved in light scattering and thermal diffusion is in progress. In the use of photoacoustic spectroscopy for quantitative analysis of solid mixtures, the likelihood of a nonlinear dependence of the photoacoustic signal on composition must be taken into account, especially when measuring in the UV
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Anal. Chem. 1981, 53, 2040-2044
more complicated in the near-IR area. Each quality control application envisaged for PAS must therefore be examined independently to assess the most suitable spectral region for analysis. Cogrinding of an absorbing material with a nonabsorbing material can considerably affect the photoacoustic response, and care must therefore be taken in sample preparation when quantitative analysis is required.
ACKNOWLEDGMENT The authors thank R. R. Ford, J. H. W. Cramp, and C. M. Keary (IC1 Corporate Laboratory), P. Hampson (IC1 Pharmaceuticals Division), S. L. Castleden (Imperial College, London), and D. M. Spillane and G. F. Kirkbright (UMIST) for helpful discussions. LITERATURE CITED PA. signal
0
40
80
Yo propranolol F/u)
Figure 6. Corrected near-infrared photoacoustlc signal as a functton of % propranolol In unground mixtures: (a) at 2.2 pm; (b) at 1.72 pm.
region. The linear dependence found in our measurements in the near-infrared spectrum suggests that quality control measurements might most successfully be applied in this region. Spectral interferences could, however, make analysis
Rosencwaig, A. Anal. Chem. 1075, 47, 592 A. Rosencwaig, A. Opt. Commun. 1073, 7 , 305. Somoano, R. 0. Aigew. Chem., Int. Ed. Engl. 1078, 17, 238. Rosencwalg, A. Optoacoustic Spectroscopy and Detection"; Academlc Press: New York, 1977; Chapter 8. Lee, L. H. Org. Coat. flast. Chem. 1070, 40, 116. \hlong, K. Y. J . Appl. fhys. 1078, 49, 3033. Adams, M. J.; King. A. A; Kirkbrlght, G. F. Analyst (London) 1078, 101, 73. Adams, M. J.; Kirkbright, G. F. Spectrosc. Lett. 1076, 9 , 255. "The Merck Index", 9th ed.; Merck & Co. Inc.: Rahway, NJ, 1978; p 7628. Lln, J. W-p.; Dudek, L. P. Anal. Chem. 1970, 51, 1627. Fuchsman, W. H.; Silversmith, A. J. Anal. Chem. 1070, 57, 589. Rosencwaig, A.; Qersho. A. J. Appl. fhys. 1078, 47, 64. Freeman, J. J.; Friedman, R. M.; Reichard, H. S. J. fbys. Chem. 1080, 84, 315.
RECEIVED for review May 26,1981. Accepted July 30, 1981.
Precision and Accuracy of Absorption-Corrected Molecular Fluorescence Measurements by the Cell Shift Method D. R. Chrlstmann,' S. R. Crouch," and Andrew Tlmnick Department of Chemistry, Mlchlgan State Unlverslty, East Lansing, Michigan 48824
Interferences due to absorption of the exciting or fluorescence radiatlon by a fluorescence sample are corrected by the cell shift method with an accuracy of better than 2% for sample absorbances as high as 2.7 If scattered llght and reemission of the absorbed fluorescence are absent. For samples that exhiblt strong self-absorption and reemisslon, the accuracy of the absorption correctlon can be poor above a fluorophore concentration of about lo-' M. Poor accuracy of the corrected fluorescence also results if the fluorescence sample Is turbld or If the spectral bandwidths of excitatlon or emlsslon are large compared to the wldth of the interferlng sample absorption band. Theoretical conslderatlons and experiments show that the precision of the corrected fluorescence is aiways poorer than the precision of the raw fluorescence, but only by a factor of 2 or less.
In a recent article, Novak (I) introduced the cell shift method for correcting inner filter effects in right-angle fluo'Present address: Boeing Aerospace 42-29, Seattle, WA 98124.
Co.,P.O. Box 3999, M/S
0003-2700/81/0353-2040$01.25/0
rometry. The method is so named because it involves shifting the sample cell in a spectrofluorometerto change the effective pathlength through which the exciting and fluorescence radiation must travel. Fluorescence intensities are measured at three cell positions. From these measurements corrections for absorption of the exciting and fluorescence beams are computed. The cell shift method offers several advantages over other absorption correction procedures in the literature (2-5). Direct measurement of the sample absorbance at the excitation and emission wavelengths is not required. A spectrofluorometer is, therefore, the only measurement device that is needed, and errors arising from the use of a spectrophotometer are avoided (I, 6). Commercial fluorescence instruments are suitable for the method after only minor modifications (6). The method has also been automated (7), resulting in a simpler calibration procedure, reduced measurement time, and reduced errors due to cell positioning. The cell shift method has been used to correct for selfabsorption and to improve the accuracy of solvent fluorescence subtraction in fluorescence emission spectra (I). Matrix absorption interferences in a fluorometric assay for aluminum have also been corrected with the technique (7). Although these applications have been successful, several factors that 0 1981 American Chemical Society
