In the Laboratory
Are Fluorescence Quantum Yields So Tricky to Measure? A Demonstration Using Familiar Stationery Products Suzanne Fery-Forgues* and Dominique Lavabre Laboratoire des Interactions Moléculaires Réactivité Chimique et Photochimique, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex, France; *
[email protected] Among experimental techniques that have demonstrated their usefulness to a vast number of disciplines is fluorescence spectroscopy. It is now extensively used in fields as varied as chemistry, physics, biology, and medical sciences, where it has proven to be a sensitive and specific method that also displays real-time capability. The efficiency of the fluorescence process is measured by the quantum yield. This parameter is of major importance. Not only is it a physical characteristic of a substance in specified conditions, but it is ultimately involved in the calculation of quenching-rate constants, energy transfer, lasing ability, and radiative and nonradiative rate constants, from which the whole photophysical behavior can be deduced. Although excellent books and reviews dealing with practical aspects of fluorescence can be found, to our knowledge very few of them (1–3) explicitly describe the procedure for the determination of quantum yield. It is not then surprising that the quantum yields given in the literature sometimes differ from one author to another for identical experiments, which makes some people reluctant to consider fluorescence spectroscopy as a reliable quantitative tool. This paper presents a routine procedure for measuring quantum yields in solution, underlining the stumbling blocks that students might encounter. By definition, the fluorescence quantum yield ΦF expresses the proportion of excited molecules that deactivate by emitting a fluorescence photon. It is the ratio of the number of emitted photons to the number of absorbed photons per time unit: Φ F = No. of emitted photons/ No. of absorbed photons (1)
It is therefore understandable that the fluorescence quantum yield is directly related to the radiative (kr) and nonradiative (knr) rate constants of deactivation by the relationship ΦF = kr /(kr + knr )
(2)
The measurement of absolute quantum yield is critical and requires special equipment. It is necessary to know with precision the amount of exciting light received by the sample. The measurements are normally done by using scattering agents and integrating spheres or actinometers to calibrate the system (2). Note that other techniques like calorimetry may also be used to determine absolute fluorescence quantum yields (4 ). For routine work, one is often satisfied with determining the relative quantum yields. The fluorescence efficiency of an unknown is then related to that of a standard by the equation ΦF(X) = (As /Ax)(Fx /Fs)(nx /ns)2 ΦF(S)
(3)
where Φ F is the fluorescence quantum yield, A is the absorbance at the excitation wavelength, F is the area under the corrected emission curve (expressed in number of photons), and n is the refractive index of the solvents used. Subscripts s and 1260
x refer to the standard and to the unknown, respectively. In this equation, absorbance A accounts for the number of absorbed photons and area F accounts for the number of emitted photons (see eq 1). Correction of the refractive indices will be discussed below. Experimental Procedures
Equipment and Materials Absorbance spectra were recorded on a Hewlett-Packard 8452A diode array spectrophotometer. Steady-state fluorescence work was performed on a Photon Technology International (PTI) Quanta Master 1 spectrofluorometer. Analytical grade absolute ethanol was obtained from Carlo Erba and used as received. Coumarin 6 and Rhodamine 101 were from Kodak. Regular yellow and pink highlighter pens (Stabilo) were purchased from the stationer. Latex examination gloves were worn to avoid staining of the hands. Sample Preparation The tips of the highlighter pens were soaked for 15 s in a small beaker containing 3 mL of absolute ethanol and the solution was filtered through paper. This was the dye stock solution. Stock solutions of Coumarin 6 and Rhodamine 101 in ethanol must be filtered through paper before use. Overview The determination of a quantum yield begins with the choice of the right standard. Then solutions of the unknown and the standard are prepared, the absorbance of each is determined, and finally the emission spectra of these solutions are recorded in order to measure the area under the curve. If different solvents are used for the standard and unknown, a correction for the refractive indices must be done. Let us detail these steps successively with the help of an example. Results and Discussion
General Procedure and Experiment 1: Determination of Fluorescence Quantum Yield of the Dye in Yellow and Pink Highlighter Pens Choice of a Standard Nowadays, on most spectrofluorometers, the signal correction is appropriate for the intensity of the exciting light as well as for the response of the monochromators and photomultiplier, all of which vary according to the wavelength. However, it is always advantageous to choose a standard with absorption and emission bands close to those of the unknown and to excite both compounds at the same wavelength. The characteristics of suitable standards are listed in the literature (see for example refs 3 and 5). Note that very few standards exist for the red and near infrared region.
Journal of Chemical Education • Vol. 76 No. 9 September 1999 • JChemEd.chem.wisc.edu
In the Laboratory
Figure 1. Absorption spectra of the highlighter pen yellow dye and coumarin 6 in ethanol. Solutions were respectively diluted 14 and 10 times for fluorescence measurement.
Figure 2. Emission spectra of the highlighter pen yellow dye and coumarin 6 in ethanol. Excitation wavelength: 420 nm; excitation bandpass: 2 nm; emission bandpass: 2 nm. Full line: corrected spectra; dotted lines: corresponding uncorrected spectra.
In the case of the yellow highlighter pen, an ethanolic solution of the dye showed absorption and emission maxima at 434 and 490 nm, respectively. Coumarin 6 in ethanol may be chosen as a standard (6 ) because its absorption and emission spectra widely overlap those of the yellow pen dye (Figs. 1 and 2). Absorbance Measurement The same procedure is used for measuring the absorbance of the unknown and the standard solutions. Absorbance must be measured with a UV–vis spectrophotometer at the wavelength that will be used later for excitation (7 ). This wavelength may differ from that of the absorption maximum. The absorbance measurement is more precise when taken on a plateau than on a sharp slope of the spectrum, except when a diode array spectrophotometer is used. In our example, we chose to excite at 420 nm. This wavelength corresponds to high absorption for both the unknown and the standard (Fig. 1). It is possible to excite at lower wavelengths; but exciting at wavelengths higher than 430 nm will not yield the full emission spectrum of the yellow dye, which begins at 435 nm. The ideal absorbance for fluorescence measurements lies between 0.05 and 0.04. When absorbance is above 0.05,
the emission intensity can no longer be assumed proportional to the concentration of the analyte (see below). On the other hand, if the absorbance is too low, impurities from the medium may become important with respect to the amount of analyte. In no case should an absorbance around 0.05–0.04 be measured directly on the spectrophotometer, because most apparatuses lack precision in this range. A simple and reliable procedure is the following. The product is dissolved, the absorbance of the solution is adjusted to around 0.5 and carefully recorded, and then the solution is diluted by a factor of 10. Setting the Spectrofluorometer The slits and gain are adjusted so that a satisfactory signal (strong enough but not saturated) is obtained when using the solution with a higher emission intensity (coumarin 6 in our example; see Fig. 2). The excitation bandwidth is kept small (8), since excitation is assumed to be monochromatic. This setting remains unchanged until the end of the experiment, so the spectra of the standard and unknown are comparable. Measurements are carried out in one session, to avoid any drift in the spectrofluorometer setting. If numerous samples are to be measured, the spectrum of the standard must be repeated from time to time. Measurement of Area under Emission Spectra The entire corrected emission spectra of the standard and unknown are successively recorded. It must be ascertained that the baseline returns to zero in the red region. The spectra are then converted to number of photons (if necessary) and integrated by using the mathematical functions of the spectrofluorometer software. (We assume that the fluorometer has all those functions, which nowadays is a minimum criterion when choosing an apparatus). To show how important the emission correction devices are, we measured the quantum yield of our product with and without correction. It was 6% lower by integrating the noncorrected spectra (Fig. 2). Determination of Refractive Indices When light passes from one medium to another, part of it is lost because of reflection, which depends upon the difference between the refractive indices of the two media. Internal reflections within the cell can also occur. Therefore, a correction must be introduced when the standard and the unknown are used in different solvents. Usually the refractive indices can be taken from a chemistry handbook (9). If necessary, the refractive index is determined using a refractometer, at the same temperature used for recording the fluorescence spectra. In our case, since ethanol was used for both the unknown and the standard solutions, the refractive index ratio was equal to 1. Calculations The data are shown in Table 1. The calculation performed by using eq 3 resulted in a quantum yield of 0.19 ± 0.02 for the yellow dye. Replicate measurements must be repeated from different solutions. Table 1. Results for the Yellow Dye Sample Coumarin 6 Yellow dye a From
ΦF
n D20
Aλex
F (area)
0.0476
1.4943 × 10
8
0.0474
3.7071 × 10
7
1.3611
a
0.78b
1.3611
a
0.19
ref 9. b From ref 6.
JChemEd.chem.wisc.edu • Vol. 76 No. 9 September 1999 • Journal of Chemical Education
1261
In the Laboratory
TLC and fluorescence spectroscopy demonstrated that a single dye was present in the highlighter pen. It was tentatively identified as a coumarin derivative. Quantum yields much higher than 0.19 can be found for that class of compounds in solution, but the manufacturer’s goal obviously was to provide a fast dye, highly fluorescent when dry. The fluorescence quantum yield of the highlighter pen pink dye was determined following the same procedure. The absorption and emission maxima were 532 and 555 nm, respectively. The compound was excited at 510 nm, using Rhodamine 101 in ethanol as a standard (ΦF = 1) (10, 11). The fluorescence quantum yield was 0.69 ± 0.07 (Table 2). This dye was identified as a rhodamine derivative. The fluorescence quantum yield determined here is in accordance with the values usually displayed by this class of compounds (11). Pitfalls In spite of precautions taken, numerous experimental pitfalls may distort the evaluation of the quantum yield. ERROR IN THE ABSORBED INTENSITY. By definition, the intensity of light absorbed by compound X in a solution (IA(X)) is an exponential function of absorbance: I a(X) = I0(1 – 10{A tot) A X /A tot (4) where I0 is the intensity of the incident beam and Atot and AX refer to the total absorbance of the solution and of that related to compound X, respectively. If X is the only absorbing species in the solution, eq 4 reduces to: (5) I a = I0(1 – 10{A ) Since the fluorescence intensity I F varies linearly with the absorbed intensity I a, IF = k I a (6) IF also varies exponentially with absorbance: IF = k I 0(1 – 10{A ) (7) This can be rewritten as IF = k I 0(1 – 10-εlc ) (8) where ε is the molar absorptivity, l is the path length, and c is the concentration of analyte. This implies that fluorescence intensity varies nonlinearly with the concentration of analyte. However, a linear dependence may be assumed between the two parameters as long as absorbance is < 0.05 (see Fig. 3).
Figure 3. Plot of fluorescence intensity vs absorbance for k I0 = 1. Solid line: “real” fluorescence, calculated from eq 6; dashed line: approximation made when fluorescence is assumed to be proportional to absorption.
1262
Table 2. Results for the Pink Dye Sample Rhodamine 101 Pink dye a From
ΦF
n D20
Aλex
F (area)
0.0456
1.9156 × 10
8
0.0487
1.4109 × 10
8
a
1.00b
a
0.69
1.3611 1.3611
ref 9. b From refs 10 and 11.
Figure 4. Light pathway in cuvettes containing (a) diluted and (b) very concentrated solutions. I 0 = Intensity of the incident light; I F = Intensity of the emitted light.
Figure 5. Highlighter pen yellow dye in ethanol. Excitation (Exc) and emission (Em) spectra of a dilute solution (A λex = 0.050); experimental (Exp) and calculated (Calc) emission spectra of a concentrated solution (A λex = 0.159). The excitation spectrum is the same as the absorption spectrum (λem = 530 nm). For emission spectra: λex = 420 nm.
Figure 6. Highlighter pen pink dye in ethanol. Excitation (Exc) and emission (Em) spectra of a dilute solution (A λex = 0.050); experimental (Exp) and calculated (Calc) emission spectra of a concentrated solution (A λex = 0.161). The excitation spectrum is the same as the absorption spectrum (λem = 610 nm). For emission spectra λex = 510 nm.
Journal of Chemical Education • Vol. 76 No. 9 September 1999 • JChemEd.chem.wisc.edu
In the Laboratory
INNER FILTER EFFECT. Inner filter effect refers to an apparent decrease in the emission quantum yield caused by the fact that the penetration of the exciting or emitting light through the cell is hindered by strongly absorbing solutions. It is a direct consequence of both the Beer–Lambert law and the particular geometry of the spectrofluorometer. Inner filter effect originates from two causes, the pre- and postfilter effects. Pre-filter Effect. Consider a sample cell that is illuminated centrally by a beam of incident light (with intensity I0) and observed at a right angle (Fig. 4a). The emitted beam (with intensity IF) detected by the apparatus originates from the center of the cell. Concentrated solutions act as a real filter, preventing light from going through the cell (Fig. 4b). In extreme cases, light is stopped before it reaches the cell center, and then no emission is detected at all. If a curve like the one in Figure 3 were plotted for very concentrated solutions, it would show fluorescence intensity reaching a maximum and then going back to zero. Post-filter Effect: Reabsorption. Distortion of band shape may also occur as a result of reabsorption of emitted radiation. This happens when the absorption and emission spectra of a compound overlap strongly.
Experiment 2: Evidence of Inner- and Post-filter Effects The decrease in fluorescence intensity related to high absorbance is illustrated by the example of the yellow dye in Figure 5. The absorbance of the diluted solution at the excitation wavelength is 0.050, whereas the absorbance of the concentrated solution is 0.159. If fluorescence intensity were proportional to absorbance, the emission spectrum of the concentrated solution would be 0.159/0.050 or 3.18 times that of the diluted solution (calculated curve in Fig. 5). Actually, the experimental curve is found to be lower than the calculated one. This results from the error in the absorbed intensity and from the pre-filter effect. The error in the calculation of the emission area is about 11%. The post-filter effect can be nicely demonstrated with the highlighter pen pink dye (Fig. 6). In comparing Figures 5 and 6, we notice that for the yellow dye, the calculated and experimental curves have the same shape. On the contrary, for the pink dye, the spectrum of the concentrated solution is distorted in the wavelength region where reabsorption occurs (between 520 and 550 nm). Obviously, the experimental spectrum is redshifted compared to the calculated one. In the latter case, the error in the absorbance intensity, pre-filter effect, and reabsorption effect are superimposed. The problem related to inner filter effect can be partially overcome by keeping the absorbance below 0.05 at the excitation wavelength. If this is not possible, the cell holder may be modified so that the path length within the cell is reduced (12). An a posteriori suitable correction may also be satisfactorily applied (13–16 ). Experiment 3: Determination of Fluorescence Quantum Yield of Dyes in Yellow and Pink Highlighter Pens between 20 and 50 °C Because the quantum yield is the ratio between radiative deactivation and thermic deactivation, it often depends on temperature. Thermostatic control should be employed during the measurement and should always be coupled with efficient stirring of the solution. Students may become aware
Table 3. Temperature Effect upon Fluorescence Quantum Yield and Emission Maximum Wavelength
t/°C
Yellow Dye ΦF λem/nm
Pink Dye ΦF λem/nm
20
0.19
491
0.69
559
35
0.17
491
0.66
557
50
0.15
491
0.63
555
of temperature dependence by measuring the quantum yield of the yellow or pink dye while passing from 20 to 50 °C. We found that the quantum yield decreased by 21% and 9%, respectively, in these conditions (Table 3). When studying variations of the fluorescence quantum yield under conditions where the shape of the emission spectrum is not modified, as for the yellow dye, determining the fluorescence quantum yield of only one sample with a standard may be enough. For the other samples, the area under the emission spectrum is proportional to the fluorescence intensity recorded at a given wavelength. Therefore the emission intensity is measured at the same wavelength for every sample, this wavelength being chosen in a region where intensity variations are important. The relative quantum yield is then obtained as ΦF = (IF/IF(0) ) ΦF(0)
(9)
where subscript (0) refers to the sample whose fluorescence quantum yield has been determined by comparison with the standard. For the pink dye, raising the temperature induces a slight shift in wavelength, so that each measurement must be done by integrating the area under the emission curve. Note that for rigorous determination of the quantum yield, the decrease of the refractive index of the dye solutions as well as any change in absorbance with increasing temperature must be corrected. In our example, the variations in absorbance within that temperature range were small enough to be neglected.
Effects of Other Factors on Fluorescence Quantum Yield Bad Dissolution of the Product Some solutions may appear clear while the product is not totally dissolved. In that case, the absorbance of the solution increases with time. The solution must be filtered. Filtration must be performed on concentrated solutions before measuring the absorbance and before diluting. This problem may be underlined by asking the students to prepare two solutions of coumarin 6, one of which is filtered and the other not. The quantum yield of the yellow dye obtained by comparison with the unfiltered standard solution should be markedly lower. Oxygen Effect It must be verified that oxygen induces no quenching effect. If that is not the case, either the solutions should be properly degassed using the freeze-pump-thaw technique or be purged with nitrogen. Impurity Effects When the fluorescence efficiency of a compound is weak, strongly fluorescent impurities may drastically interfere with the measurement of the quantum yield. Analytes should be
JChemEd.chem.wisc.edu • Vol. 76 No. 9 September 1999 • Journal of Chemical Education
1263
In the Laboratory
as pure as possible and solvents should be of fluorescence grade and checked for spurious emission. Polarization Effects The transmission efficiency of a grating monochromator depends on the plane of polarization of light with respect to the grating grooves (17, 18). The resulting emission spectrum then differs in intensity and shape according to the state of polarization of light. This effect is important when exciting with polarized light, as is commonly done during anisotropy measurements. However, during routine nonpolarized measurements, the excitation monochromator gratings induce a slight polarization of light. This effect is weak, since most of the solutions emit depolarized light. The preferred way to overcome this difficulty is to set a polarizer at emission at the “magic angle” (54.7° from the vertical). In these conditions, the obtained signal is proportional to the total emission intensity, whatever the state of polarization (18, 19). Of course the spectra of the standard and the unknown must be recorded under the same conditions. Using polarizers decreases the intensity of the signal. Raman Scattering The Raman peak due to the solvent may appear in the emission spectrum of one of the samples. It is easy to recognize because it is a sharp triangular peak with a position that varies when changing the excitation wavelength. It may be troublesome when the sample signal is weak. In that case, the Raman peak, obtained by determining the emission spectrum of the solvent alone, must be substracted from the spectrum of the sample. Photochemical Instability The fluorometer source is intense enough to induce modifications of photochemically unstable analytes. They may react with or without the involvement of oxygen. These reactions may either decrease the fluorescence signal or amplify it according to the nature of the product formed. The presence of this problem is evidenced by variations as a function of time for the same cell. To cope with this problem, one may try reducing the excitation slits or increasing the scanning speed. Gentle stirring of the sample allows the measurement to be done on a homogeneous medium, since only a small amount of solution is reacted at a time and this is continuously mixed with fresh solution.
1264
Conclusion In the literature fluorescence quantum yields are commonly given with a 10% error. Actually, one must anticipate much larger uncertainty because their measurement may be tricky indeed, even for skilled people. However, observing the simple rules stated above should make things better for the beginner. Acknowledgments R. Nagarajan and an unknown referee are gratefully acknowledged for helpful suggestions. Literature Cited 1. Parker, C. A.; Rees, W. T. Analyst (Cambridge, UK) 1960, 85, 587–600. 2. Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991–1024. 3. Standards for Fluorescence Spectrometry; Miller, J. N., Ed.; Chapman and Hall: London, 1981. 4. Fisher, M.; Georges, J. Anal. Chim. Acta 1996, 334, 337–344. 5. Eaton, D. F. J. Photochem. Photobiol. B 1988, 2, 523–531. 6. Reynolds, G. A.; Drexhage, K. H. Opt. Commun. 1975, 13, 222. 7. It is also possible to determine fluorescence quantum yield without directly measuring absorbance. The ratio of two fluorescence intensities measured at two points of the excitation spectrum is thus related to the absorbance and to the fluorescence efficiency of the solution. See: Britten, A.; Archer-Hall, J.; Lockwood, G. Analyst (Cambridge, UK) 1978, 103, 928–936. 8. Bendig, J.; Kreysig, D.; Schoeneich, R. Z. Chem. 1979, 19, 151–152. 9. Handbook of Chemistry and Physics, 65th ed.; Weast, R. C., Ed.; CRC: Boca Raton, FL 1985. 10. Karstens, T.; Kobs, K. J. Phys. Chem. 1980, 84, 1871–1872. 11. Drexhage, K. H. J. Res. Natl. Bur. Stand. A 1976, 80A, 421–428. 12. Lutz, H. P.; Luisi, P. L. Helv. Chim. Acta 1983, 66, 1929–1935. 13. Lopez Arbeloa, I. J. Photochem. 1980, 14, 97–105. 14. Mode, V. A.; Sisson, D. H. Anal. Chem. 1974, 46, 200–203. 15. Gill, J. E. Appl. Spectrosc. 1970, 24, 588–590. 16. Rohatgi, K. K.; Singhal, G. S. Photochem. Photobiol. 1968, 7, 1–7. 17. Luminescence Spectroscopy; Lumb, M. D., Ed.; Academic: London, 1978; pp 190–192. 18. Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum: New York, 1983 ; pp 26–29. 19. Pesce, A. J.; Rosen, C. G.; Pasby, T. L. Fluorescence Spectroscopy: An Introduction for Biology and Medicine; Dekker: New York, 1971; pp 171–172.
Journal of Chemical Education • Vol. 76 No. 9 September 1999 • JChemEd.chem.wisc.edu
