3384
J. Phys. Chem. 1980, 84, 3384-3389
Scheme I
Currently with Department of Radiation Bblcgy, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan. H. Neubacher and 0. H. Schnepel, Radlat. Res., 72, 48 (1977). A. WybWk and J. Mybeck, photochem. R~~fobb/., 16,359 (1972). A. Meybeck and J. J. Windie, Photochem. Photoblol., 10, 1 (1969). G. M. Schnepei and H. Neubacher, Radlat. Envlron. Biophys., 13, 49 (1976). R. B. Johns, F. D. Looney, and D. J. Whelan, Photochem. Photobbl., 7. 65 (1962). L. J. Mittal, J. P. Mittal, and E. Hayon, Photochem. Photoblol., 18, 281 (1973). L. J. Mittal, J. P. Mittal, and E. Hayon, J. Phys. Chem., 77, 1482 (1973). 0. Leuschner, H. Jeschkeit, and 0. Losse, Photochem. Photoblol., 5, 705 (1968). E. G. Janzen, Acc. Chem. Res., 2, 279 (1969). E. G. Janzen, Top. Stareochem., 6, 177 (1971). P. Giasoe and F. A. Long, J . Phys. Chem., 64, 188 (1980). R. 0. Bates, “Determinationof pH”, Wiiey, New York, 1973, p 375. S. N. Rustgi and P. Riesz, Int. J. Radlat. Biol. Relat. Stud. phys., Chem. Med., 84, 301 (1978). L. J. SaMel, A. R. Goklfarb, and S. Waldman, J. 8/0/. Chem., 197, 285 (1952). L. J. Saidel, Arch. Biochem. Blophys., 54, 184 (1955). L. J. Saidel, Arch. Biochem. Biophys., 56, 45 (1955). S. Rustgi and P. Riesz, Int. J. Radlat. Blol. Rebt. Stud. Phys., Chem. ~ e d .38, , 325 (1978). C. Lagercrank and M. Setaka, Acta Chem. Scad., 326,619 (1974). P. D. Sullivan and E. M. Menger, Adv. Magn. Reson., 9, 1 (1977). T. Siddai, W. Stewart, and F. Knight, J. Phys. Chem., 74, 3580 (1970). W. Stewart and T. Siddal, Chem. Rev., 70, 517 (1970). S. Rustgi, A. Joshl, P. Riesz, and F. Friedberg, Int. J. Radlat. Blol. Relat. Stud. Phys., Chem. ~ e d .82, , 533 (1977). S. Rustgi and P. Riesz, Int. J. Rad/&. Biol. Relat. Stud. Phys., Chem. Med., 34, 127 (1978). F. Kerkhoff, Z . Phys., 158 595 (1960). J. H. Shulman and W. D. Compton, “Color Centers in Solids”, Macmilian,,New York, 1962, p 185. E. J. Hart and M. Anbar, “The Hydrated Electron”, Wiley-Interscience, New York, 1970 p 96. Y. Kirino and H. Taniguchi, J. Am. Chern. Soc., Q8,5089 (1976). A. L. Lehninger, “Biochemistry”, 2nd ed., Worth Publishers, New York, 1975, p 135.
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was also photolyzed and the same spectrum was observed. Experiments on the photolysis of peptides containing the aromatic residues phenylalanine, tyrosine, and tryp tophan are in progress. The results obtained with aliphatil peptides are helpful in interpreting the more comple: spectra obtained with these peptides. The spin-trapping method is expected to contribute to a better understanding of some aspects of the photochemistry of proteins and enzymes. References and Notes (1) Currently with Institut de Physique, Universit6 de Liage, 4000 Sart-Tilman par Liege I, Belgium.
Photoacoustic Calorimetry of Concentrated Fluorescent Solutions David Cahen,” Haim Garty, and Ralph S. Beckert Weizmann Institute of Science, Rehovot, Israel (Received: June 9, 1980)
Photoacoustic (PA) detection is used to determine fluorescence quantum yields of concentrated disodium fluorescein (Na2F1)and cresyl violet perchlorate (CVP) solutions. ,Use of heat dissipation spectra, which are obtained from the ratio of the corrected photoacoustic and transmission absorption spectra, is found to give the most reliable results. The presence of several species with different fluorescence characteristics,apparent from the PA data, limits the wavelength range over which the dissipation spectrum can be used to obtain quantitative information. The occurrence of PA saturation leads to erroneous results if data for only one wavelength are used; however, its effect cancels when extrapolation of heat dissipation spectra is employed. Characteristic features of PA detection of fluorescence that are noted include the fact that total fluorescence is sensed and that the technique is essentially a thin film reflectance one. Such features, which can complicate direct comparison of P A data with conventional ones, may be used advantageously in specific problems.
Introduction Photoacoustic (PA) detection is being used widely in spectroscopy, especially in cases where conventional transmission or reflectance techniques pose problems.’ Because the technique is essentially a photocalorimetric one, in that it measures that part of the radiation absorbed +
On leave from Department of Chemistry, University of Houston. 0022-3654/80/2084-3384$01 .OO/O
by the sample that is converted into heat, it can be used for the study of luminescent material^"^ and samples undergoing photoinduced chemical changes.“ll When used in this way it can complement more conventional techniques, such as fluorescence spectroscopy. Several configurations of the method have been described, namely, gaseous samples in contact with a conventional microphone,s liquid samples in contact with a 0 1980 American Chemical Society
Photoacoustic Calorlmetry of Fluorescent Solutions piezoelectric t r a n ~ d u c e rliquid ,~ samples in contact with a specially designed capacitor microphone,12and the most common one, where a liquid or solid sample is surrounded by a gas phase, which is in contact with a conventional rni~rophone~l9~ Our studies on energy transduction in biological samples, utilizing the last configuration, involve indirect detection of the heat produced by the sample which has the advrmtage that contributions from direct, light-induced, volunne changed2 are minimall3 This allows an easier interpretation of the results in terms of enthalpy changes.13J4 Using B simple theoretical interpretation of the PA signal from photoactive samples,l0 we arrived at procedures to reference and calibrate PA signals in terms of the energy used for photoactivity. The present study was undertaken to check these procedures, using samples whose photoactivity is well known, i.e., fluorescent solutions. This use of the PA method is similar to conventional calorimetry, for which uncomplicated experimental setups have been reported, and which has been used for the determination of absolute fluorescence quantum yields and for the study of photochemical reactions.15J6 Because of the somewhat inefficient method of detection in our PA experiment (involving several energy transduction steps), only concentrated solutions of our samples, disodium fluorescein (Na2F1)and cresyl violet perchlorate (CVP), had sufficiently high optical absorptions to give satisfactory signal-to-noise ratios. (S/N values obtained in our experimental setup were, however, superior to those obtained on a PAR 6001 commercial instrument, for these kinds of ~amp1es.l~) This use of concentrated solutions led to some results not E!xpected from data available for dilute solutions. In the case of CVP, dimer formation limited the range of wavelengths over which quantiative information, on quantum yields, could be obtained. For Na2Fl the rather high excitation light intensity led to decreased fluoirescence not observed in normal fluorescence spectra. Here, too, the range of wavelengths over which quantitative information could be extracted was significantly smaller than that expected for dilute solutions. Taking into account these restrictions, we could obtain satisfactory quantum yield values using PA data. In this way we coulcl confirm the validity and accuracy of an extrapolation method (vide infra) to energetically calibrate the PA signals, especially if the optical absorption characteristics of the PA sample and the thickness of the photoacousticdly active layer are known. In addition, our results (a) confirm the possibility to extract calorimetric data from samples that show PA saturation,lJ7J8 (b) indicate the usefulnes8 of the method to observe small changes in fluorescence characteristics of highly fluorescent samples, (c) show how PA data help to identify wavelength regions of relatively constant photoactivity, and (d) enable detection of photoac tivity and aggregation in optically dense solutions. Experimental Section PA data were obtained from 25-30-pL samples, surrounded by air, at room temperature, situated in cells previously described,lg using lock-in detection of the microphone (Knowles Electronics Co., BT1753) signal. Although only small chunges in phase angle occurred over the wavelength ranges of interest, the vector sum of the in-phase and quadrature signal was measured, thus maximizing the PA. signal. PA excitation came from a 450-W Xe arc lamp, whose output was chopped mechanically, and passed through a Baiisch and Lomb Hi-intensity monochromator and cutoff' filter, to suppress second-order radiation. A 500-nm blaze (1350 grooves/mm) grating and 4-8-nm bandwidth slits were used. The modulation fre-
The Journal of Physical Chemistry, Vol. 84, No. 25, 1980 3385
quency was measured by an analog frequency meter, as well as by observing the reference signal on an oscilloscope. Transmission spectra were measured on Cary 118 and 1605 spectrophotometers, using 0.1-mm optical pathlength Nippon Sekei quartz cells, to enable measurement of the very solutions used in the PA experiment. Background signals were negligible and sample luminescence did not influence the transmission spectra. Fluorescence measurements were obtained by using a Perkin-Elmer MPF 44A spectrophotometer, equipped with a correction unit (concentrated rhodamine B was used for correcting Na2Fl spectra; the lamp + excitation monochromator spectrum was measured directly, by the emission photomultiplier, to enable correction of the CVP spectra). Fluorescence spectra were all taken in the front surface mode with the sample, in the ID.1-mm cell, at 45' with respect to the emission and excitation slits (5-nm bandwidth, unless otherwise stated)^. Background signals (from solvents only) were negligible. Light intensities were measured by using a Yellow Springs Industries radiometer, and a home-built photometer, equipped with a Si photocell, calibrated by ferrioxalate actiinometry. In the PA experiments the excitation light intensities were between 50 and 150 W/m2 and in the transmission and fluorescence experiments they were
