Visible

Anal. Chem. 2001, 73, 4958-4963

Separation of Scattering and Absorption Contributions in UV/Visible Spectra of Resonant Systems Norberto Micali,†,‡ Francesco Mallamace,‡,§ Mariangela Castriciano,⊥ Andrea Romeo,⊥ and Luigi Monsu´ Scolaro*,‡,⊥

Istituto di Tecniche Spettroscopiche, ITS-CNR, Messina, Italy, INFM, Unita` di Messina, Messina, Italy, Dipartimento di Fisica, Universita` di Messina, Messina, Italy, and Dipartimento di Chimica Inorganica, Chimica Analitica, e Chimica Fisica, Universita` di Messina and Istituto di Chimica dei Prodotti Naturali (ICTPN-CNR), Sezione di Messina, Messina, Italy

Resonance light scattering (RLS) is a phenomenon due to an enhancement of the scattered light in close proximity to an absorption band. The effect is easily detectable in the case of strongly absorbing chromophores, which are able to interact, thus leading to large aggregates (Pasternack,R. F.; Collings, P. J. Science 1995, 269, 935). The measurement of absorption spectra from solutions containing such resonant systems can lead to misleading results. In this paper, a simple method is described to obtain absorption spectra of aggregated species with a fairly good correction of the scattering component. The RLS spectrum, obtained using a common spectrofluorimeter, is correlated to the extinction spectrum of the same sample, allowing for an estimation of the scattering contribution to the total extinction spectrum. The method has been successfully applied both on real samples containing aggregated chromophores, such as porphyrins, chlorophyll a and gold colloids, and by simulating extinction spectra. Aggregation of small molecules is an area of active research because of the many implications in different fields of chemistry, biology and physics.1 A particular interest has been addressed to systems in which arrays of chromophores are structurally organized. These supramolecular assemblies exhibit physicochemical properties, which are important from a fundamental point of view and are suitable for possible technological applications, for example, materials with enhanced nonlinear optical susceptibilities.2 In biological systems, ordered assemblies of bacteriochlorophylls are found in the light-harvesting complexes LH-I and LHII of purple bacteria3-6 and in the chlorosomes of green * To whom correspondence should be addressed. Fax: +39-090-393756. E.mail: [email protected]. † Istituto di Tecniche Spettroscopiche. ‡ Unita ` di Messina. § Dipartimento di Fisica, Universita ` di Messina. ⊥ Dipartimento di Chimica Inorganica, Chimica Analitica, e Chimica Fisica, Universita` di Messina and Istituto di Chimica dei Prodotti Naturali. (1) Lehn, J. M. In Supramolecular Chemistry; VCH: Weinheim, 1995. (2) Spano, F. C.; Mukamel, S. Phys. Rev. A 1989, 40, 5783. (3) McDermott, G.; Prince, S. M.; Freer, A. A.; HawthornthwaiteLawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. W. Nature 1995, 374, 517. (4) Karrash, S.; Bullough, P. A.; Ghosh, R. EMBO J. 1995, 14, 631.

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photosynthetic bacteria.7 Several mimetic aggregates based on synthetic porphyrins have been tailored as light harvesting systems for artificial photosynthesis and as molecular devices.8,9 Recently, nano- and micrometric porphyrin wheels, potentially mimicking these natural arrays, have been obtained by simple solvent evaporation.10,11 A different problem is the formation of supramolecular assemblies of charged porphyrins on oppositely charged polymeric matrixes, for example DNA,12-14 RNA,15,16 and polypeptides.17-21 In the absence of a templating support, the buildup of highly structured H- or J-aggregates, corresponding to the limiting cases of parallel units aligned face-to-face or edgeto-edge, respectively, has been reported.22-30 J-aggregates are also common for a series of aromatic compounds, that is, cyanine (5) Cogdell, R. J.; Fyfe, P. K.; Barrett, S. J.; Prince, S. M.; Freer, A. A.; Isaacs, N. W.; McGlynn, P.; Hunter, C. N. Photosynth. Res. 1996, 48, 55. (6) Pullerits, T.; Sundstrom, V. Acc. Chem. Res. 1996, 29, 381. (7) Blankenship, R. E.; Olson, E. J. M.; Miller, M. In Anoxygenic Photosynthetic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer, C. E., Eds.; Kluwer Academic Publishers: Dordrecht, 1995, 399. (8) Ward, M. D. Chem. Soc. Rev. 1997, 26, 365. (9) Hayashi, T.; Ogoshi, H. Chem. Soc. Rev. 1997, 26, 355. (10) Schenning, A. P. H. J.; Benneker, F. B. G.; Geurts, H. P. M.; Liu, X. Y.; Nolte, R. J. M. J. Am. Chem. Soc. 1996, 118, 8549. (11) Hofkens, J.; Latterini, L.; Vanoppen, P.; Faes, H.; Jeuris, K.; Defeyter, S.; Kerimo, J.; Barbara, P. F.; Deschryver, F. C.; Rowan, A. E.; Nolte, R. J. M. J. Phys. Chem. B 1997, 101, 10588. (12) Gibbs, E. J.; Tinoco, I., Jr.; Maestre, M. F.; Ellinas, P. A.; Pasternack, R. F. Biochem. Biophys. Res. Commun. 1988, 157, 350. (13) Pasternack, R. F.; Bustamante, C.; Collings, P. J.; Giannetto, A.; Gibbs, E. J. J. Am. Chem. Soc. 1993, 115, 5393. (14) Mukundan, N. E.; Petho, G.; Dixon, D. W.; Kim, M. S.; Marzilli, L. G. Inorg. Chem. 1994, 33, 4676. (15) Pasternack, R. F.; Brigandi, R. A.; Abrams, M. J.; Williams, A. P.; Gibbs, E. J. Inorg. Chem. 1990, 29, 4483. (16) Pasternack, R. F.; Gurrieri, S.; Lauceri, R.; Purrello, R. Inorg. Chim. Acta 1996, 246, 7. (17) Pancoska, P.; Urbanova, M.; Bednarova, L.; Vacek, K.; Panschenko, V. Z.; Vasiliev, S.; Malon, P.; Kral, M. Chem. Phys. 1990, 147, 401. (18) Nezu, T.; Ebert, G. Biopolymers 1991, 31, 1257. (19) Gibbs, E. J.; Pasternack, R. F. J. Inorg. Organomet Polym. 1993, 3, 77. (20) Purrello, R.; Monsu` Scolaro, L.; Bellacchio, E.; Gurrieri, S.; Romeo, A. Inorg. Chem. 1998, 37, 3647. (21) Purrello, R.; Monsu` Scolaro, L.; Lauceri, R.; Gurrieri, S.; Romeo, A. J. Am. Chem. Soc. 1998, 120, 12353. (22) Ohno, O.; Kaizu, Y.; Kobayashi, H. J. Chem. Phys. 1993, 99, 4128. (23) Ribo`, J. M.; Crusats, J.; Farrera, J. A.; Valero M. L. J. Chem. Soc., Chem. Commun. 1994, 681. (24) Pasternack, R. F.; Schaefer, K. F.; Hambright, P. Inorg. Chem. 1994, 33, 2062. (25) Akins, D. L.; Zhu, H.-R.; Guo, C. J. Phys. Chem. 1994, 98, 3612. 10.1021/ac010379n CCC: $20.00

© 2001 American Chemical Society Published on Web 09/12/2001

dyes,31,32 xanthenes,33 and polycyclic aromatic hydrocarbons.34 In the case of J-aggregates derived from cyanine dyes, important applications as photographic sensitizers35 have been developed as a result of their giant absorption cross section and exciton delocalization capability.36,37 The complexity of these systems has demanded the development and application of many experimental techniques for investigating their structural and dynamical properties. Recently, an unusual light scattering technique, termed resonance light scattering (RLS), has been proposed to investigate the formation of aggregated chromophores in complex systems.38 The RLS effect has been reported in a large variety of systems, spanning from small chromophores (lycopene),39 semiconductors,40 porphyrin24,29,30 and chlorophyll a41 aggregates, and dye-protein42-44 and dye-nucleic acid12,13,16,45 aggregates. The RLS effect arises from an enhancement of the scattered light intensity in close proximity to an absorption band. The occurrence of this phenomenon is related to a strong electronic coupling between adjacent chromophores, the size and the geometry of the resulting aggregate, and intense molar absorbance of the monomeric constituents.38 A quantum mechanical model for RLS based on exciton-coupling theory has been recently developed.46 A consequence of resonance-enhanced scattering is that the “absorption spectrum”, as measured by a conventional spectrophotometer, is the sum of two extinction components due to absorption and scattering, respectively. A clear example of this phenomenon has been reported for the aggregate of trans-H2Pagg, which, in the presence of nucleic acids, “absorbs” at 450 nm. Indeed, this spectral feature has a large component due to scattering that appears as extinction in the UV/vis spectra.13 Another classic example of extinction is given by colloidal gold. The absorption spectrum of a sol colloidal ruby red suspension of gold particles (radii in the range 26-100 Å) exhibits a band at ∼520 nm as a result of surface plasma resonant absorption.47 On (26) Maiti, N. C.; Ravikanth, M.; Mazumdar, S.; Periasamy, N. J. Phys. Chem. 1995, 99, 17192. (27) Akins, D. L.; Zhu, H.-R.; Guo, C. J. Phys. Chem. 1996, 100, 5420. (28) Barber, D. C.; Freitag-Beeston, R. A.; Whitten, D. G. J. Phys. Chem. 1991, 95, 4074. (29) Rubires, R.; Crusats, J.; El-Hachemi, Z.; Jaramillo, T.; Lopez, M.; Valls, E.; Farrera, J. A.; Ribo`, J. M. New J. Chem. 1999, 189. (30) Collings, P. J.; Gibbs, E. J.; Starr, T. E.; Vafek, O.; Yee, C.; Pomerance, L. A.; Pasternack, R. F. J. Phys. Chem. B 1999, 103, 8474. (31) Mobius, D. Acc. Chem. Res. 1981, 14, 63. (32) Nakahara, H.; Fukuda, K.; Mobius, D.; Kuhn, H. J. Phys. Chem. 1986, 90, 6144. (33) Valdes-Aguilera, O.; Neckers, D. C. Acc. Chem. Res. 1989, 22, 171. (34) Hessemann, J. J. Am. Chem. Soc. 1980, 102, 2176. (35) Daehne, S. Photogr. Sci. Eng. 1979, 23, 219. (36) Fidder, H.; Terpstra, J.; Wiersma, D. A. J. Chem. Phys. 1991, 94, 6895. (37) Moll, J.; Daehne, S.; Durrant, J. R.; Wiersma, D. A. J. Chem. Phys. 1995, 102, 6362. (38) Pasternack, R. F.; Collings, P. J. Science 1995, 269, 935. (39) Anglister, J.; Lin, S. H.; Blankenship, R. E. Chem. Phys. Lett. 1979, 65, 50. (40) Gurioli, M.; Bogani, F.; Ceccherini, S.; Vinattieri, A. Colocci, M. J. Opt. Soc. Am. B 1996, 13, 1232. (41) de Paula, J. C.; Robblee, J. H.; Pasternack, R. F. Biophys. J. 1995, 68, 335. (42) Ma, C. Q.; Li, K. A.; Tong, S. Y. Anal. Biochem. 1996, 239, 86. (43) Ma, C. Q.; Li, K. A.; Tong, S. Y. Fresenius’ J. Anal. Chem. 1997, 357, 915. (44) Borissevitch, I. E.; Tominaga, T. T.; Imasato, H.; Tabak, M. Anal. Chim. Acta 1997, 343, 281. (45) Huang, C. Z.; Li, K. A.; Tong, S. Y. Anal. Chem. 1997, 69, 514. (46) Parkash, J.; Robblee, J. H.; Agnew, J.; Gibbs, E. J.; Collings, P. J.; Pasternack, R. F.; dePaula, J. C. Biophys. J. 1998, 74, 2089.

increasing the size of the particles (>100 Å), the spectral feature becomes broader and moves to longer wavelengths (up to 680 nm). Because of the nature of the scatterers, that is, chromophores with large extinction coefficients, absorbance can constitute a severe problem in the analysis and interpretation of RLS data. Recently, Collings et al. proposed a method to perform the correction and to dissect the absorption and the scattering components.30 Because UV/vis spectroscopy is extensively used as a tool to investigate the electronic properties of aggregated chromophores, we thought it worthwhile to further address the importance of recognizing and correcting the experimental spectra from extinction due to resonant scattering. Here we report on a fairly simple method (it uses a common spectrofluorimeter) exploiting the RLS technique to obtain absorption spectra of aggregated species with a fairly good correction of the scattering component. The method of Collings et al. requires the exact knowledge of the structure factor of the scatterer and has to take into account polarization effects. The present method, even if it represents a rough approximation, can give a good estimate of the amount of resonant scattering in an extinction spectrum. The method has been applied to colloidal gold suspensions, J-aggregates of meso-tetrakis(4-sulfonatophenyl)porphine (H2TPPS44-)5,9-12 and aggregated chlorophyll a.41 EXPERIMENTAL SECTION Chemicals and Preparation of Aggregated Samples. HAuCl4 (Strem) and meso-tetrakis(4-sulfonatophenyl)porphine (tetrasodium salt, Aldrich) were used as received. Chlorophyll a was a gift of Prof. A. Agostiano (Bari, Italy). An aggregated sample in a 9:1 solution of formamide and pH 7 phosphate buffer was prepared according to a literature method.41 Colloidal aqueous gold suspensions (sol state) were prepared following a literature method.48 A solution of HAuCl4 (0.005% w/w, 750 mL) was heated at 100 °C for 12 min, and then 37.5 mL of a solution of citric acid (0.15% w/w) was added. The boiling mixture became blue after 2 min, and after 3 min, it turned reddish purple. Dynamic light scattering measurements indicate that the particles have an average hydrodynamic radius of 40 ( 1 nm. J-aggregates of the diacid porphyrin H2TPPS44- were prepared by adding 2.9 M sodium chloride to a 5 µM solution of H2TPPS44(tetrasodium salt) at pH 2.8 (HCl) and 298 K. The complete aggregation was checked through UV/vis spectroscopy by monitoring the increase of the 489-nm Soret band of the J-aggregated porphyrin.22-27 Spermine-induced J-aggregates of H2TPPS44- were prepared by adding a known volume of a concentrated stock solution of polyamine (Sigma) to a solution of porphyrin (3-5 µM) in 10 mM citrate buffer, pH 2.5.49 Instrumentation. UV/vis spectra were measured on a HewlettPackard model HP 8453 diode array spectrophotometer. Resonancelight-scattering experiments were performed on a Jasco model FP-750 spectrofluorimeter equipped with a Hamamatsu R928 PMT using a synchronous scan protocol with a right angle geometry.38 (47) Kreibig, U.; Vollmer, M. Optical Properties of Metal Clusters; Springer: New York, 1995. (48) Frens, G. Nature 1973, 241, 20. (49) Monsu` Scolaro, L.; Romeo, A. Unpublished results.

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Our approach to the problem of separating the absorption and the scattering components is based on the possibility of factoring the function Q(λ,θ,φ) in the product Q(λ)F(θ,φ). In general, this procedure is not practicable, being the scattering angular profile strictly connected to the wavelength of the radiation through the particle size of the medium. In the particular case in which the turbidity of the medium is due to resonant scattering, the function Q(λ,θ,φ) is not zero only in a restricted range of wavelengths, for which we can assume that the angular and the wavelength dependence are decoupled.

S(λ) )

∫∫ Q(λ,θ,φ) dθ dφ ) Q(λ) ∫∫ F(θ,φ) dθ dφ

(2)

Figure 1. Scattering geometry for a particle illuminated by an incident plane wave.

This assumption implies that the scattering profile is dependent only on the wavelength in the spectral range in which the scattering is fairly different from zero. Under this hypothesis, it is possible to choose a factor, FF, that accounts for the integral ∫∫ F(θ,φ) dθ dφ normalized as

Q(λ) RESULTS AND DISCUSSION General Theory. When radiation passes through an absorbing and turbid medium, its extinction, E(λ) ) (λ)c (where (λ) is the extinction coefficient and c the concentration), is the sum of both absorbance, A(λ), and scattering, S(λ):50

(S(λ) + A(λ))Lt ) E(λ)Lt

(1)

where Lt is the optical path length of the radiation in the medium. The scattering, S, is the integral over all the angles of the scattering amplitude, Q. This latter quantity is function of wavelength λ and polarization of the radiation and the observation angles θ and φ (where θ is the scattering angle, and φ is the azimuthal angle, which define the scattering plane, Figure 1).51 The function Q(λ,θ,φ) is also strongly dependent on the nature of the medium. The knowledge of size and shape distributions of the scatterers, geometry of polarization, and refractive index (real and imaginary) of the particles allows for an evaluation of the integrated value of S. An experimental method to measure absorbance in sensibly scattering media is based on the use of an integration sphere. This device is generally able to collect only the light that propagates in the forward direction, leading to a modest correction of the total extinction. Another method, which is selective only for the absorbance, is the photoacoustic technique, in which the detection is based on the “acoustic noise” due to the heating of the medium at the absorption wavelengths. This nonconventional method, even if not sensitive to the scattering, is difficult to calibrate in absorbance units, and the obtained spectra contain only qualitative information (i.e., it is possible to identify absorption features in an extinction measurement).52 (50) Bohren, C. F.; Particles; John (51) Bohren, C. F.; Particles; John (52) Bohren, C. F.; Particles; John

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Huffman, D. R. Absorption and Scattering of Light by Small Wiley: New York, 1983; Chapter 11, p 287. Huffman, D. R. Absorption and Scattering of Light by Small Wiley: New York, 1983, Chapter 3, p 61. Huffman, D. R. Absorption and Scattering of Light by Small Wiley: New York, 1983, Chapter 11, p 319.

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∫∫ F(θ,φ) dθ dφ ) Q

90(λ)FF

(3)

where Q90 is the scattering at θ ) 90° and φ ) 0°. The intensity I(λ), as measured by a spectrofluorimeter with a right angle geometry and a synchronous scanning of both excitation and emission monochromators (the usual setup for RLS spectra), is related to the Q90(λ) through the transfer function T(λ) (characteristic of the two monochromators and the detector) and the initial intensity I0(λ)

I(λ) ) I0(λ)T(λ)Q90(λ)10-E(λ)Ls + c

(4)

where Ls is the average path (effective) of the radiation in the scattering process and c is a constant term that accounts for incoherent contributions (e.g., dark counting of the detector). The average path Ls can be made very similar to the geometric one Lt by using cells with a diaphragm, or alternatively, it can be properly calibrated. It should be noted that we are using the extinction and not the absorbance for the calculation of the scattered light intensity that is collected by the detector. This is due to the fact that both absorbance and scattering contribute to the intensity loss. At this point, S(λ) can be calculated from I(λ).

S(λ) )

FF(I(λ) - c)10E(λ)Ls I0(λ)T(λ)

(5)

And finally, the extinction E(λ) is calculated from

E(λ) ) A(λ) + S(λ) ) A(λ) +

FF(I(λ) - c)10E(λ)Ls (6) I0(λ)T(λ)

This equation can be rearranged and expanded considering only the first-order terms, that is, for E(λ) < 0.4. Under the assumption that the transfer function T(λ) and the initial intensity I0(λ) are nearly constant and can be approximated to an average

value in the investigated wavelength range, eq 7 follows.

E(λ) - A(λ) ) a(I(λ) - c) ) aI(λ) + b

(7)

where a ) FF/I0(λ)T(λ) To obtain the absorption A(λ) from the experimental extinction E(λ) and the intensity measured on the spectrofluorimeter I(λ), the constant terms a and c have to be determined and fixed. This determination can be easily performed by a linear fitting of E(λ) vs I(λ) in a wavelength range in which the extinction is almost entirely due to scattering (i.e., A(λ) ) 0, a condition that is easily met in resonant systems), according to the equation E(λ) ) S(λ) ) a I(λ) + b. Therefore, in the spectral range in which I(λ) * 0, the contribution of the absorption A(λ) to the total extinction will be given by

A(λ) ) E(λ) - aI(λ) - b

(8)

Under the same assumptions, the scattering intensity corrected for the extinction is

Q90(λ) ∝ (aI(λ) - b)(1 + 2.3026E(λ)Ls)

(9)

It should be noted that (i) only this quantity is dependent on the average path Ls, and (ii) because of the limitation of eq 6 to only the linear terms in A and E, it is possible to use interchangably the absorption or the extinction in the determination of I(λ) by eq 4. Simulations of Extinction Spectra and Separation into Absorption and Scattering Components. Extinction spectra, E(λ), were simulated by adding two Gaussian components for the absorption, A(λ), and the scattering, S(λ), spectra, respectively. The simulations were performed by varying the relative amplitudes, A0 and S0, the center of the bands, λmax, and the line widths, w. The RLS spectra, I(λ), were obtained from the theoretical scattering component, by modulating the intensity with the absorption (i.e. I(λ) ) S(λ) × 10(-Ls.Abs(λ)). To determine the coefficients a and b of eq 7, a linear regression was performed of I(λ) vs E(λ) in a region in which absorption is almost negligible. Application of eqs 8 and 9 allows the recalculation of the absorption and scattering components. Figure 2 reports a typical procedure for an extinction spectrum composed of a Gaussian absorption (λmax ) 50, A0 ) 0.1, and w ) 5) and a scattering (λmax ) 50, S0 ) 0.1, and w ) 25) component. A linear fitting procedure was applied in the region 70-100, giving a ) 0.1362 ( 0.0013 and b ) -0.01045 ( 0.00073 (R ) 0.99856). It is easy to realize that a good separation of the components can be achieved when (i) the separation between the two peaks is larger of the biggest line width, w, or (ii) the line width of the scattering component is much larger than that of the absorption. We want to outline the importance of choosing the proper spectral range to perform the analysis, because it is necessary to avoid the absorption feature, which leads to severe distortion, or to go far away introducing, in such a case, noise in the data. Separation of Absorbance and Scattering in Real Cases. To perform the correction, the average path length Ls of the light beam into the sample has to be first determined. As previously cited, it is possible to use special fluorescence cells with geo-

Figure 2. Simulated extinction spectrum and calculated absorption component (see text): (A) simulated extinction (‚‚‚‚), absorption (s) and scattering (- - -), (B) calculated RLS profile, I(λ), modulated for the absorption, (C) calculated (‚‚‚‚) and simulated (s) absorption component, (D) diagram of E(λ) vs I(λ) in the region of 70-100 nm, with the result of the linear fit (solid line).

metrically designed diaphragms. In our case, we used the method reported by Collings et al., based on a fitting procedure of data obtained with solutions containing latex particles and potassium dichromate.30 The application of this procedure gave Ls ) 0.9 cm for our spectrofluorimeter. Our method has been tested with different aggregated systems. Figure 3 shows the extinction spectrum (A) and the resonance light scattering profile (B) of a sample of colloidal gold. The involvement of absorption and scattering in generating the broad band at 520 nm in the extinction spectrum is clearly pointed out by quite a large RLS component at 570 nm, that is, in the long wavelength tail in the feature of spectrum A. Accordingly, a diagram of the extinction vs the RLS intensity of the sample in a preresonance range (600-700 nm) is linear. A linear regression of these data gave a ) (3.40 ( 0.01) × 10-4 and b ) (1.56 ( 0.11) × 10-2 (R ) 0.991). The correction is shown in trace C of Figure 3. The same method was applied on colloidal solutions containing aggregated porphyrins. At pH < 1 or in the presence of a high salt concentration, the diprotonated form of the anionic H2TPPS44porphyrin forms J-aggregates, characterized by a narrow and redshifted absorption band at 489 nm. 22-27 The RLS spectra evidence Analytical Chemistry, Vol. 73, No. 20, October 15, 2001

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Figure 5. Correction of the UV/vis spectrum of an aggregated sample of H4TPPS42- in the presence of spermine ([H4TPPS42-] ) 5 µM, [spermine] ) 100 µM, citrate buffer 10 mM, pH 2.5).

Figure 3. (A) UV/vis spectrum of a colloidal gold suspension; (B) RLS profile of the same sample; (C) correction of the spectrum (see text).

Figure 4. Correction of the UV/vis spectrum of an aggregated sample of H4TPPS42- ([H4TPPS42-] ) 5 µM, pH 2.8, [NaCl] ) 2.9 M).

a strong feature centered at ∼500 nm and in agreement with the presence of extensively aggregated species.24 The spectrum after the correction is reported in Figure 4C. A slight reduction of the line width of the 489 nm band corresponding to the J-aggregate is evident. On the contrary, the correction does not affect the Soret band of the diacid H4TPPS42- porphyrin (434 nm). This result is quite interesting, because usually the square root of the aggregation number in J-aggregates is reported as proportional to the line width of the red-shifted band.53 A slight shift in the position of this band, together with an excitation-dependent fluorescence emission has also been considered as indicative of the presence of a polydispersed population of aggregates.26 It is important at this point to note that the corrected spectra showed in Figure 4 are very similar to those reported by Collings et al. on the same system. Apart from the above-reported limitations, the main (53) Knapp, E. W. Chem. Phys. 1984, 85, 73.

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Figure 6. Correction of the UV/vis spectrum of an aggregated sample of chlorophyll a in a 9:1 solution of formamide and phosphate buffer ([Chl a] ) 5 µM, pH 7.0, phosphate buffer 75 mM).

advantage of the simple method here presented is that it does not require the exact knowledge of the angular dependence of the scattering (structure factor), which has to be determined through more sophisticated laser light-scattering equipment.30 The correction of the resonant scattering component is much more pronounced in the case of spermine-induced J-aggregates of H2TPPS44-.49 The RLS profile is characterized by a large scattered intensity with a peak at 500 nm, almost matching the broad and long tail of the corresponding extinction spectrum (Figure 5). The linear correlation of the two data sets in a region between 500 and 600 nm is fairly linear and allows performance of the correction, which is reported in Figure 5C. The effect of the enhanced scattering in close proximity to the absorption band is evident. The last example is the UV/vis spectrum of aggregated chlorophyll a in organic solvent/water mixtures. As reported in the literature, chlorophyll a is able to aggregate in a 9:1 mixture of formamide and phosphate buffer, eventually leading to an aggregated species, Chl469, which exhibits a strong RLS feature at 469 nm and an UV/vis band at 453 nm.41 An inspection of the UV/vis spectra reveals the presence of a broad tail in the main Soret band at 453 nm. The regression analysis was performed in the range of 460-530 nm, and the corrected spectrum is reported in Figure 6. CONCLUSIONS Even if a rigorous separation of scattering from absorption cannot neglect the scattering structure factor of the system and the instrumental wavelength dependence of the scattered intensity,30 the method reported in this paper can give a fairly good

estimation of the two components. Furthermore, it is easy to perform, because it requires the use of instruments commonly available in a research laboratory (a spectrophotometer and a spectrofluorimeter). The occurrence of assemblies of chromophores, which give resonance-enhanced light scattering, is not uncommon in many supramolecular systems having biological relevance and technological applications. Because UV/vis spectra are often exploited to obtain information on the geometrical arrangement of interacting chromophores,54,55 it is important to (54) McRae, E. G.; Kasha, M. In Physical Processes in Radiation Biology; Augenstein, R., Mason, L., Rosenberg, B., Eds.; Academic Press: New York, 1964; pp. 23-42. (55) McRae, E. G.; Kasha, M. J. Chem. Phys. 1958, 28, 721.

take this phenomenon into account in order to avoid a misleading interpretation of the experimental spectroscopic parameters. ACKNOWLEDGMENT We thank the Ministero dell’ Universita´ e della Ricerca Scientifica e Tecnologica (MURST), Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale, Cofinanziamento 200001, INFM/PRA, and CNR for funding this work. We gratefully acknowledge useful discussions with Prof. R. F. Pasternack and Prof. P. J. Collings. Received for review April 3, 2001. Accepted July 16, 2001. AC010379N

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