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; *sff@gdp.ups-tlse.fr
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 ex-
presses 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 quan-
tum 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 Journal of Chemical Education • Vol. 76 No. 9 September 1999 • JChemEd.chem.wisc.edu
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 correc-
tion 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 charac-
teristics 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.

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 spectro-
photometer 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,
JChemEd.chem.wisc.edu • Vol. 76 No. 9 September 1999 • Journal of Chemical Education
In the Laboratory
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 absor-
bance 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 inte-
grated by using the mathematical functions of the spectrof-
luorometer 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 emis-
sion 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 refrac-
tometer, 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 Aλex F (area) n D20 ΦF
Coumarin 6 0.0476 1.4943 × 10 8
1.3611 a
0.78b
Yellow dye 0.0474 3.7071 × 10 7
1.3611 a
0.19
a From ref 9. b From ref 6.
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 In the Laboratory

TLC and fluorescence spectroscopy demonstrated that a Table 2. Results for the Pink Dye
single dye was present in the highlighter pen. It was tenta- Sample Aλex F (area) n D20 ΦF
tively identified as a coumarin derivative. Quantum yields
Rhodamine 101 0.0456 1.9156 × 10 8
1.3611 a
1.00b
much higher than 0.19 can be found for that class of compounds
in solution, but the manufacturer’s goal obviously was to Pink dye 0.0487 1.4109 × 10 8
1.3611 a
0.69
a From ref 9. b From refs 10 and 11.
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
Figure 4. Light pathway in cuvettes containing (a) diluted and (b)
In spite of precautions taken, numerous experimental very concentrated solutions. I 0 = Intensity of the incident light; I F =
pitfalls may distort the evaluation of the quantum yield. Intensity of the emitted light.
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:
I a = I0(1 – 10{A ) (5)
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) Figure 5. Highlighter pen yellow dye in ethanol. Excitation (Exc)
and emission (Em) spectra of a dilute solution (A λex = 0.050);
where ε is the molar absorptivity, l is the path length, and c experimental (Exp) and calculated (Calc) emission spectra of a
is the concentration of analyte. This implies that fluorescence concentrated solution (A λex = 0.159). The excitation spectrum is the
intensity varies nonlinearly with the concentration of analyte. same as the absorption spectrum (λem = 530 nm). For emission
However, a linear dependence may be assumed between the spectra: λex = 420 nm.
two parameters as long as absorbance is < 0.05 (see Fig. 3).

Figure 6. Highlighter pen pink dye in ethanol. Excitation (Exc) and

Figure 3. Plot of fluorescence intensity vs absorbance for k I0 = 1. emission (Em) spectra of a dilute solution (A λex = 0.050); experimental
Solid line: “real” fluorescence, calculated from eq 6; dashed (Exp) and calculated (Calc) emission spectra of a concentrated solu-
line: approximation made when fluorescence is assumed to be tion (A λex = 0.161). The excitation spectrum is the same as the absorp-
proportional to absorption. tion spectrum (λem = 610 nm). For emission spectra λex = 510 nm.

1262 Journal of Chemical Education • Vol. 76 No. 9 September 1999 • JChemEd.chem.wisc.edu


INNER FILTER EFFECT. Inner filter effect refers to an ap-
parent 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 post-
filter 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 ex-
treme 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 exci-
tation 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 con-
centrated 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 calcu-
lated 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 exci-
tation 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 satisfac-
torily 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 radia-
tive 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
JChemEd.chem.wisc.edu • Vol. 76 No. 9 September 1999 • Journal of Chemical Education
In the Laboratory
Table 3. Temperature Effect upon
Fluorescence Quantum Yield and
Emission Maximum Wavelength
Yellow Dye Pink Dye
t/°C
ΦF λem/nm 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 spec-
trum 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 in-
tensity 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 mea-
suring 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
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 mea-
surements, 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 propor-
tional 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 am-
plify 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 prob-
lem, 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 Journal of Chemical Education • Vol. 76 No. 9 September 1999 • JChemEd.chem.wisc.edu
Conclusion
In the literature fluorescence quantum yields are com-
monly 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 ac-
knowledged for helpful suggestions.
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