The fraction of the number of quanta absorbed by a molecule that are emitted as fluorescence
is termed the fluorescence quantum yield. Its determination provides information con-
cerning radiationless processes in molecules and, for example, the determination of the
potential of fluorophores in assays. Quantum yields are measured either on a relative basis
with reference to a standard or by using an absolute method. Both methods have been
reviewed by Demas and Crosbyl and by Bridges2
Absolute quantum yields were measured by Weber and Teale3 by using the dipolar
scattering of monochromatic light from glycogen solutions as a standard unit of quantum
yield. A comparison was then made with the fluorescence from a solution with the same
apparent absorbance for the excitation light. Other methods for determining absolute
quantum yields include photoacoustic spectroscopy4 and calorimetric r n e t h ~ d s . ~
The most widely used method of determining quantum yields is by the relative method
and the quantum yield of the unknown, Qx,is calculated according to the following equation:
QX -- Q R *
- - EX_ I-R & ..
Ax * E R * I , ' ni
where QR is the quantum yield of the standard, A is the absorbance of the solution, E is the
corrected emission intensity, I is the relative intensity of the exciting light and n is the
average refractive index of the solution. Subscripts R and X refer to the reference and
unknown compound, respectively. When a Rhodamine quantum-corrected reference system
is used, the relative intensity of the excitation light a t different wavelengths is taken to be
unity, which simplifies equation (1) to
The following factors can affect the measurement of relative quantum yields : polarisation;
refractive index changes ; re-absorption of the emission ; internal reflection ; variation of
optical density with band width ; and calibration errors between ultraviolet absorption and
fluorescence spectrometers.
The use of an integrating sphere6 largely overcomes the errors associated with polarisation
and refractive index changes. Working at very dilute absorbances or extrapolating to zero
absorbance eliminates errors from re-absorption and internal reflection. The errors associ-
ated with measuring the absorbance values are based upon the difference in characteristics
between an ultraviolet - visible absorption spectrometer and a fluorescence spectrometer.
* Presented at the Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectro-
scopy, Atlantic City, N J, USA, March, 1983.
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1068 RHYS WILLIAMS et al. : RELATIVE QUANTUM YIELDS USING Analyst, Vol. 108
Unless two identical monochromators are used errors may arise from two sources. Firstly,
the effect of spectral band width on the shape of the ultraviolet absorption spectrum, hence
absorbance value ; the shape of the fluorescence excitation spectrum, hence relative intensity.
For example, the ultraviolet absorption spectrum of pyrene in hexane for two different slit
widths is shown in Fig. 1. This illustrates the change in absorbance values and peak ratios,
which occur as the spectral band width is changed. Unless the fluorescence spectrometer
has an identical monochromator the excitation spectra will also be different.'
Published on 01 January 1983 on http://pubs.rsc.org | doi:10.1039/AN9830801067
0.8
0.6
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C
D
0
n
i Oa4
a
0.2
n
520 260 300 340 380 220 260 300 340 380
Wavelengthlnm
Fig. 1. Ultraviolet absorption spectra of
pyrene in hexane with (a) 2-nm band width and
(b) 4-nm band width.
at 20 nm as recommended by Melhuish.13
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100
From excitation To emission
80 monochromator monochromator
%
0
2 60
2
Q)
20 Plane
mirror
1070 RHYS WILLIAMS et al. : RELATIVE QUANTUM YIELDS USING Analyst, VoZ. 108
response graph. All of these procedures were performed on the Model 3600 data
station.
The Model LS-5 luminescence spectrometer was used as a single-beam ultraviolet - visible
absorption spectrometer by placing a mirror at the sample focus to reflect the excitation
light into the emission monochromator. The light needs to be heavily attenuated with a
wire-mesh gauze to prevent overloading of the sample photomultiplier. A 10-mm path-
length cuvette was placed in the reflected beam (Fig. 4) and by synchronously scanning both
monochromators at the same wavelength, a transmission spectrum was obtained. By
dividing a sample spectrum by the solvent spectrum the transmission spectrum of the solute
was obtained. The latter was converted to an absorbance spectrum. Fig. 5 compares the
Published on 01 January 1983 on http://pubs.rsc.org | doi:10.1039/AN9830801067
absorbance spectrum of pyrene in hexane, as measured on the Model LS-5, with the excita-
tion spectrum. Table I compares the ratios of the main peaks as measured on the ultra-
violet absorption spectrometer and on the Model LS-5. Excellent agreement between the
ultraviolet absorption and fluorescence excitation spectra is observed. In addition, the
absorbance value falls midway between the values observed on the ultraviolet spectrometer
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O.*
I
0.6 -
8
0
n
5 0.4
n
a
0.2 -
Wavelengthlnm
Fig. 5. (a)The absorbance spectrum and (b)
the excitation spectrum of pyrene in hexane as
measured on the Model LS-5.
calibration range of fluorescence emission ‘uemws concentration. The solutions were not
de-gassed before measurement. The emission spectra were measured using a 2.5-nm band
width and corrected using the correction graph in Fig. 2 and the areas under the corrected
spectra calculated. The quantum yields were calculated according to equation (2) and the
results by this method compared with some literature values, which are given in Table 11.
The reproducibility of measuring and calculating the quantum efficiency of the same solution
is within *3-5%.
Published on 01 January 1983 on http://pubs.rsc.org | doi:10.1039/AN9830801067
TABLE
I1
ROOMTEMPERATURE FLUORESCENCE QUANTUM YIELDS
The following abbreviations are used : 9,10-DPA, 9,lO-diphenylanthracene ; 9-MA,
9-methylanthracene; TPB, tetraphenylbutadiene; QS, quinine sulphate; C, cyclohexane;
Downloaded by Indian Institute of Science on 11 August 2011
The method proposed for calculating relative fluorescence quantum yield was found to
produce results that compare favourably with those previously published. The discrepancy
between the observed and literature value for fluorene is probably the result of a relatively
low value observed by Weber and Teale3 because Dawson and Windsor15 observed a value
of 0.68. The main advantage of this technique is the ability to measure absorbance values
on a fluorescence spectrometer thus eliminating errors associated with the use of an absorption
spectrometer. The use of a desk-top computer greatly simplifies the experiment and routine
quantum yield measurements are easily performed by automating the control of the instru-
ment and data calculation.
References
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2. Bridges, J . W., in Miller, J. N., Editor, “Standards in Fluorescence Spectroscopy” Chapman and
Hall, London, 1981, p. 68.
3. Weber, G., and Teale, F. W. J., Trans. Faraday Soc., 1957, 53, 646.
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5. Olmsted, J., J . Phys. Chem., 1979, 83, 2581.
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