Determinations of Fluorescence Quantum Yields
The Journal of Physical Chemistry, Vol. 83,
No. 20, 1979 2581
Calorimetric Determinations of Absolute Fluorescence Quantum Yields John Olmsted, I11 Department of Chetnlstry, California State University, Fullerton, Fullerton, California 92634 (Recelved April 2, 1979) Publication costs assisted by the Research Corporatlon
The absolute fluorescence quantum yields, &, of a number of highly fluorescent organic dyes and aromatic compounds have been measured in various solvents, utilizing a calorimetric technique in which the heating rates under monochromatic irradiation are compared for the fluorescent solution and for an inert reference absorber. The technique yields the absolute fluorescenceyield for air-saturated solutions with an accuracy of f0.02. The compound/solvent systems studied provide reference fluorescence values of 0.50 or greater for solvents of refractive index varying between 1.33 and 1.50 and at excitation wavelengths ranging from 366 to 633 nm.
Introduction The fluorescence quantum yield of a molecule is one of the key photophysical quantities that are amenable to direct experimental determination. Because of its dependence on the fundamental unimolecular rate constants for excited state decay, the fluorescence yield is of substantial importance in numerous areas of chemistry. As these areas have been described in some detai1,l only a few will be briefly mentioned here: theory of excited state decay, mechanistic organic photochemistry, fluorescence probing of biological molecules, laser development. Because of formidable difficulties attendant on absolute determinations of fluorescence yields, fluorescence is almost always studied relative to some standard substancea2 The reliability of such relative determinations is then limited both by the accuracy of the standard yield value and by the confidence that can be placed on the comparison technique. Both of these factors have proved to be restrictive in the past. On one hand, very few absolute standards have been assigned reliable yield values. Several years ago, the critical review of Demas and Crosby indicated only two systems (quinine bisulfate in 1.0 N sulfuric acid, 0.546; fluorescein in 0.1 N sodium hydroxide, 0.90) whose fluorescenceyields were probably reliable to within 5%. Since that time, these two standards have been further strengthened3f4and two others have been proposed: 9,lO-diphenylanthracene in deoxygenated cyclohexane, 0.95,536and cresyl violet in methanol, 0.54.7 The list remains strikingly short. On the other hand, comparative techniques require close attention to factors such as excitation wavelength, emission spectra, reabsorbance, and the refractive index of the solution. Ideally, in a comparative measurement one would like to have identical absorbance throughout the excitation bandwidth, closely similar wavelength distributions of the fluorescence spectra, negligible reabsorbance of the emission, and identical refractive indices of the two solutions being compared. With so few reliable fluorescence standards available, it is seldom possible to achieve all of these features; hence it has usually been necessary to introduce correction factors (e.g., emission response calibration curves, n2 refractive index corrections) which are themselves subject to a degree of uncertainty. As a result, although relative fluorescence quantum yields can be measured relatively easily, they can seldom be reported with better than about 10% reliability. Rectification of this difficulty requires a whole series of absolute standards encompassing as wide as possible a range of wavelength regions and solvent refractive index and thereby permitting the experimenter to select a standard which closely matches the characteristics of the 0022-365417912083-258 1$01.OO/O
system being studied. We have recently developed a relatively uncomplicated calorimetric technique which is capable of giving fluorescence quantum yields of highly fluorescent solutions with good precision and reproduci b i l i t ~ . ~Using , ~ this technique, we have determined the fluorescence yields of a series of potential standard substances in solvents of varying refractive index. The results of those determinations are reported here.
Experimental Section Materials. The following chemicals were used as received from the supplier: quinine bisulfate (Eastman), coumarin 1 (Eastman Laser Grade), perylene (AldrichGold Label, 99+ %), acridine yellow (Aldrich), cresyl violet (Exciton), 1,1’,3,3,3’,3’-hexamethylindodicarbocyanine iodide (HlDC) (Eastman). Rhodamine 6G showed no impurities upon TLC but was nonetheless recrystallized from an ethanol-ether mixture prior to use. The recrystallized material had the same quantum yield as an unrecrystallized sample. Fluorescein, which had been purified by the method of Orndorff and Hemmer,8was a generous gift from Dr. D. Magde and was used as received. The sample of 16,17-dihydroxyviolanthronebis(hexanoate) ester was kindly provided by Dr. Arthur Mohan, American Cyanamide Co. It was used without further purification and after TLC with a variety of solvents showed only a single spot. “Perfect absorber” reference compounds for all solvents except water were azulene or tetraphenylcyclopentadienone (Aldrich),which were used as received. Reagent grade potassium dichromate was used as reference in aqueous solvent. All of the solvents except water were fractionally distilled using a Vigreux column. In all cases, a single distillation yielded solvent which showed neither absorbing nor emitting impurities throughout the spectral range used in these experiments. Water was glass distilled. Spectral Measurements. Absorbance spectra and matching were carried out on a Cary 15 spectrophotometer by using matched 5-cm cells. Fluorescence spectra and oxygen quenching factors were determined with an Aminco-Bowman spectrofluorimeter equipped with an RCA 4840 photomultiplier tube. Calorimetry. The photomicrocalorimeter was basically the same as described previ~usly.~ Two different Dewar sample cells were used for these experiments. For most of the determinations, a 3.00-mL volume cell of 1 X 1 cm square-walled Pyrex tubing, silvered only on its upper half, was used. For irradiations at 633 nm, where the photon flux from the source was low and the light beam crosssection small, a cylindrical cell of 0.5-cm diameter and 0.6-mL volume was used. Three different light sources 0 1979 American Chemical Society
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The Journal of Physical Chemistry, Vol. 83, No. 20, 7979
John Olmsted, 111
TABLE I : Calorimetrically Determined Fluorescence Quantum Yields useful range,a hfluorrb compd
solvent
quinine bisulfate fluorescein rhodamin 6 G rhodamin 6 G acridine yellow rhodamin 6 G rhodamin 6 G cresyl violet cresyl violet methylene blue coumarin ~f coumarin I acridine yellow rhodamin 6 G rhodamin 6 G cresyl violet cresyl violet methylene blue per y 1en e perylene violanthroneh violanthrone violanthrone coumarin I perylene per y 1ene violanthrone violanthrone violanthrone HID0 HIDC
1.0 N H,SO, 0.1 N NaOH H,O H,O met han ol methanol methanol methanol methanol methanol ethanol ethanol ethanol ethanol ethanol ethanol ethanol ethanol cyclohexane cyclohexane cyclohexane cyclohexane cyclohexane toluene toluene toluene toluene toluene toluene Me,SO Me,SO
,
nm
nm
I N IIair
280-380 435-515 470-555 470-555 410-480 470-555 470-555 570-635 570-635 570-680 32O-41Og 3 20-4 1Og 410-480 470-555 470-555 5 10-6 35 5 10-635 580-690 350-445g 35 0-445g 465-600 465-600 465-600 3 20-395' 350-4 50g 350-450' 470-610 470-610 470-610 575-675 575-675
456.5 532.5 565 565 517 573 573 623 623 710 460 460 517 573 573 623 623 710 465 465 611 611 611 418 470 470 630 630 630 680 680
1.00 1.00 1.00 1.00
1.10 1.01 1.01 1.00 1.00
1.00 1.10 1.10 1.08 1.00 1.00 1.00 1.00 1.00 1.25 1.25 1.09 1.09 1.09 1.16 1.32 1.32 1.03, 1.03, 1.03, 1.00 1.00
excitn A,' nm 366 488 488 546 436 488 546 546 578 633 366 404 436 488 546 546 578 633 404 436 488 546 578 366 404 436 488 546 578 578 633
@p(i0.02) 0.55 (0.546) 0.90e(0.90) 0.76 0.81 0.57 0.86 0.80 0.55 0.55 0.03, 0.64 0.64 0.47 0.88 0.88 0.50 0.51 0.04 0.73 0.75 0.88' 0.76' 0.84' 0.56 0.71 0.75 0.88 0.88 0.84 0.31' 0.26'
a Wavelength range over which extinction coefficient is at least 10% of its maximum value. Mean wavelength of fluorescence emission spectrum. Hg line excitation, except 488 nm (Ar ion laser) and 633 nm (He-Ne laser). dFluorescence quantum yield for air-saturated solutions. e Undergoes photodecomposition with a quantum yield of ca. lo-,. 4-MethylN,N-dimethyl-7-aminocoumarin. Onset of second singlet absorption. 16,17-Dihydroxyviolanthronebislhexanoate) ester. E Fluorescence quantum yield appears t o be wavelength dependent; not recommended as standards. 1,1',3,3,3',3'hexamethylindodicarbocyanine iodide. J
were used. Mercury line excitation was accomplished with an Illumination Industries Inc. 100-W short-arc lamp and housing, whose output was filtered by the appropriate Baird-Atomic bandpass optical interference filter (10-nm bandpass, transmittance less than 0.01% in blocking regions). Excitation at 488 nm utilized a Spectra-Physics Model 164 argon-ion laser operated at 0.20-W output power. At 633 nm, a 1-mW helium-neon laser served as the excitation source. In all cases, the temperature sensor was a Sargent S-81620 low range thermistor used in conjunction with a Sargent thermistor bridge and a 1-mV strip-chart recorder; this system had a sensitivity of deg/division. For the 633-nm runs, the bridge output was amplified by a Hewlett-Packard 425A microvolt-ammeter which enhanced the sensitivity 10 times. The experimental procedure consisted of a sequence of fixed-time irradiations of fixed-volume aliquots of inert reference, pure solvent, and fluorescent standard in turn. Reference and standard had matched absorbances (CO.100 for 1-cm path length) at the irradiation wavelength. The mean rate of heating of each solution was determined by averaging heating rates, corrected as needed for thermal drift, for a set of a t least four different irradiations. Typical values for pure solvent heating rates were deg/s. deg/s and for the inert reference samples, 2 X From these mean heating rates, the fluorescence quantum yield was calculated from the e q ~ a t i o n ~ > ~
where r, s, and b refer to the inert reference, fluorescence
sample, and solvent blank, respectively. Other Measurements. Values for air quenching of fluorescence were obtained by comparing the fluorescence intensities of three aliquots of dilute solutions, one of which was air-saturated while the others were saturated with oxygen or nitrogen by bubbling a solvent-saturated stream of the gas through the fluorescence cuvet. Mean values of the emission wavelength were calculated from published corrected emission spectra when available or from uncorrected fluorescence spectra taken in our laboratory otherwise.
Results and Discussion Quantum yields of fluorescence determined in this work are reported in Table I along with other parameters related to the use of these compounds as fluorescence standards. The first column of the table lists the compound and the second, the solvent used. In the third column is given the useful range of excitation wavelengths. This is the wavelength region over which the extinction coefficient is 10% or more of the emax, such that reference solutions of measurable optical density could be prepared without causing substantial reabsorption effects on the fluorescence. For coumarin I and perylene, the onset of the second singlet absorption band is given as the lower limit of the useful range, inasmuch as it is not known whether or not the fluorescence quantum yield remains independent of wavelength when S2is excited. The fourth column tabulates the mean emission wavelength of the compound, which was used in computing the fluorescence quantum yields. Column five gives the fluorescence intensity ratios between nitrogen- and air-
Determinations of Fluorescence Quantum Yields
saturated samples, allowing conversion from the airquenched quantum yields given in the table to intrinsic unimolecular fluorescence yields in the absence of bimolecular quenching. Column six lists the excitation wavelengths at which the compound was studied in this work, and column seven gives the mean fluorescence quantum yields at each wavelength for air-saturated solutions as determined by the calorimetric technique. The quoted limits of accuracy for the fluorescence quantum yields are k0.02, based on several considerations. First, the reproducibility of repetitive measurements lay within these limits. Second, when measurements could be made at more than one excitation wavelength, the results at different wavelengths agreed to within these limits, with two exceptions as noted in the table. Third, for those compounds for which there is in the literature a consensus value for the fluorescence quantum yield, the values reported here are in excellent agreement with that consensus value. Fourth, analysis of the precision of the experimental determinations as outlined below leads to predicted error limits of this magnitude. Sources of error which must be considered in this technique are errors in the mean excitation and emission wavelength, reabsorption and reemission of emitted light, mismatches of the optical densities of the reference and sample solutions, and inaccuracies in determining the solution heating rates. Of these possibilities, only the latter two are significant under our conditions. The absorption wavelength is known to about 0.1 nm, since line sources and efficient interference filters were utilized. The emission wavelength is known with lesser accuracy, since it must be obtained by integration of measured emission spectra which may not be properly corrected for detector wavelength response. However, comparison of corrected and uncorrected emission spectra for those systems for which corrected spectra are available (e.g., perylene in cyclohexane) showed differences of less than 5 nm in the mean emission wavelength, or about 1%of the mean value. Reabsorption is rendered negligible in these experiments by keeping solution optical densities at the level of 0.1 or lower. Optical densities were matched with a pair of 5-cm path length matched quartz cells and a Cary 15 spectrophotometer. The cell match was within the noise level of the instrument, which was f0.002 absorbance units at an optical density of 0.5. Since the mercury lines in particular had significant line widths, and in many cases the solution absorbances were varying by as much as 0.02 absorbance units per nanometer, slight mismatches of total solution absorbance (over the entire line width) could be occurring even though matches within instrument noise were achieved at the mean absorption wavelength. Such mismatches are difficult to estimate quantitatively, inasmuch as they depend not only on the rate of change of solution absorbances with wavelength but also on the line width and degree of asymmetry of the exciting lines. In no case should they exceed 1-2 % , and in favorable cases (laser excitation, reference and sample absorbancesvarying similarly with wavelength) they should be within instrument noise. Determination of the slopes of the heating curves involved both a baseline measurement and a measurement of chart deflection resulting from a fixed time of irradiation. Baseline slopes were typically 0-3 divisions/min, whereas heating rates during irradiation were of the order of 60-90 divisions/min for reference solutions and 20-80 divisions/min for the fluorescent samples. These slopes could be determined with a reproducibility of 1-2 divi-
The Journal of Physical Chemistry, Vol. 83, No. 20, 1979 2583
sionslmin, yielding the quoted uncertainty in the fluorescence yields of f0.02. Of the compounds listed in Table I, several are not recommended for quantum yield standards. Methylene blue, whether in methanol or ethanol solvent, has a quite low fluorescence yield which is only stated with an error limit of about 50%. It is included in the table since it has been suggested as a standard2and since there is a paucity of standards in the far-red spectral region. Fluorescein undergoes photochemistry with a decomposition yield of the order of 10-6.10As we have confirmed, this is sufficient to cause measurable changes in its fluorescence intensity during the time period of a typical fluorescence quantum yield determination, and it is therefore to be avoided as a standard substance unless special precautions are taken always to use fresh solutions. Violanthrone in cyclohexane and HlDC in dimethyl sulfoxide both gave variations in quantum yield with excitation wavelength that appear to be outside of the range of experimental error. These variations may represent solvent-solute interactions, association phenomena, or the like which cause variations in the fluorescence. These systems should not be used as standards without further study. For aqueous solutions, quinine bisulfate remains an ideal choice at shorter wavelengths, and rhodamin 6G appears promising in the vicinity of 500 nm. The latter compound must be used at low concentrations to avoid aggregation, but its fluorescence is relatively insensitive to pH variations.ll For the intermediate and red wavelength regions, no aqueous standard is yet available, particularly in view of the susceptibility of fluorescein to photodecomposition. Methanolic solutions, however, have refractive indices only slightly different from water (1.331 for methanol and 1.328 for water); hence methanol reference solutions could readily be used for aqueous measurements and vice versa. The set of reference compounds, quinine sulfate-1.0 N H2S04,acridine yellow-methanol, rhodamin GGmethanol, and cresyl violet-methanol, thus covers effectively the entire excitation wavelength region from 280 to 635 nm. Similarly, the compounds listed in Table I for ethanol (RI = 1.362), cyclohexane (1.429), and toluene (1.497) effectively encompass the entire excitation wavelength range from about 320 nm to at least 600 nm. Since solvent refractive indices from 1.33 to 1.50 are represented, standard solutions can be selected that will minimize the necessity for refractive index corrections for almost any solvent system of interest; and in any case, the refractive index of the sample can be bracketed by those of two standards from the table. Apart from the fluorescein and quinine sulfate standards, few of the compounds listed in Table I have been the subject of prior quantitative investigations. Drexhage has recommended rhodamin 6G as a standard, reporting its quantum yield to be 0.95 relative to 0.90 for fluorescein and solvent-independent to within 10% The values reported here are somewhat lower than this, but in view of the instability of fluorescein and the possible inaccuracy of refractive index corrections alluded to by Drexhage, the two sets of results seem to be substantially in agreement. Berlman has reported a yield of 0.94 for perylene in air-free cyclohexane and an oxygen quenching factor of 1.30,12 which give an air-saturated quantum yield of 0.725,in good agreement with the 0.74 value found in this work, The fluorescence yields reported here demonstrate that it is generally unjustified to assume that fluorescence parameters are minimally influenced by solvent. Coumarin I, for example, has a yield of 0.64 in ethanol and 0.56 in toluene. In cyclohexane, its absorption spectrum is
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The Journal of Physical Chemistry, Vol. 83, No. 20, 1979
John Olmsted, I11
qualitatively different and it undergoes phot~degradation.~~ commercial purity (cresyl violet, perylene, coumarin I), or Most of the other compounds studied in this work show were purified before use (fluorescein, rhodamin 6G), or solvent variations in their fluorescence quantum yields of showed no major impurities upon TLC (violanthrone, acridine yellow). 10-20%, indicating that solvent-solute interactions play a measurable role in modifying unimolecular decay conA further important experimental consideration is that stants for excited singlet electronic states. for compounds showing small Stokes’ displacements of the A question of some interest is whether a compound emission and absorption spectra, low optical densities are which is a “perfect emitter” exists. In order for the a necessity to avoid effects due to reabsorption and fluorescence quantum yield to approach 1.0, it is necessary reemission. With the exception of quinine bisulfate and for the radiative rate constant to exceed the nonradiative coumarin I, all of the standard compounds in Table I are rate constants by a factor of 100 or more. It has been subject to such effects. It was for this reason that solution optical densities were kept below 0.1 at the wavelength of suggested that 9,lO-diphenylanthracene is such an emitter,12 but the best value for its fluorescence yield is maximum extinction coefficient and, in using these 0.95.516 Of the compounds studied in this work, all of which compounds as standards, similarly low concentrations were selected because of their intrinsically high fluoresshould be employed. cence efficiencies, only perylene displays an intrinsic Acknowledgment, This research was supported in part unimolecular fluorescence efficiency in excess of 0.90, and by a grant from the donors of the Petroleum Research its yield is 0.96 in toluene. I t would appear that those Fund, administered by the American Chemical Society, factors which cause rapid radiative decay, at least for and by a Cottrell Research Grant from the Research organic dye molecules and aromatic hydrocarbons such as Corporation. Mr. Raymond Villalobos, an undergraduate were studied in this work, also tend to make nonradiative research student at California State University, Fullerton, processes (intersystem crossing and/or internal conversion) assisted in collection of some of the data. relatively efficient. It should be emphasized that although the fluorescence yields reported in this work are probably reliable to f0.02, References and Notes these values can be used with confidence only if proper (1) G. A. Crosby, J. N. Demas, and J. B. Callls, J. Res. Natl. Bur. Stand., care is taken with experimental conditions. As mentioned Sect. A , 76, 561 (1972). (2) J. N. Demas and 0. A. Crosbv, J . Phys. Chem., 75. 991 (1971). . . above, fluorescence quantum yields show solvent de(3) B. Gelernt, A. Findeisen, A. Stein, and 2. A. Poole, J. Chem. SOC., pendences of up to 20%; thus values determined in one Faraday Trans. 2 , 70, 939 (1974). solvent cannot be applied for another. Many organic dyes (4) J. H. Brannon and D. Magde, J . Phys. Chem., 82, 705 (1978). (5) M. Mardelll and J. Olmsted, 111, J . Photochem., 7 , 377 (1977). are subject to aggregation phenomena, particularly in polar (6) W. R. Ware and W. Rothman, Chem. Phys. Lett., 39, 449 (1976). solvents; hence the values reported here, which are for (7) D. Made, J. H. Brannon, T. L. Cremers, and J. Olmsted, 111, J. Phys. solutions having optical densities less than 0.1 (and Chem., 83, 693 (1979). (8) W. R. Orndorff and A. J. Hemmer, J . Am. Chem. Soc., 49, 1272 therefore for concentrations in the M range), should (1927). not be used for solutions of higher concentrations without (9) P. G. Seybold, M. Gouterman, and J. Callis, Photochem. Photobiol., first ascertaining that the higher concentration solutions 9, 229 (1969). accurately obey Beer’s law. All of the systems studied in (10) J. Weber, Phys. Len. A , 45, 35 (1973). (11) K. H.Drexhage, J. Res. Natl. Bur. Stand., Sect. A , 80, 421 (1976). this work did follow Beer’s law at the low optical densities (12) I. B. Berlman, “Handbook of Fluorescence Spectra of Aromatic used. Purity of the standard should also receive careful Molecules”, 2nd ed, Academic Press, New York, 1971. (13) J. Olmsted, 111, unpublished observations. attention. Compounds used in this work were of very high
