Absolute Quantum Yields Using an Integrating Sphere

Download PDF August 16, 2022


FP-8500 Spectrofluorometer
FP-8500 Spectrofluorometer


Different molecular and environmental conditions not only effect whether a molecule will fluoresce or not, but can also determine the intensity of the emitted fluorescence radiation. A molecule’s efficiency to fluoresce is described by its quantum yield and is defined as the ratio of the number of photons absorbed to the number of photons emitted by a sample. There are two methods for measuring the fluorescence quantum yield: the absolute method and the relative method. The absolute method directly obtains the quantum yield by detecting all sample fluorescence through the use of an integrating sphere.  The relative method compares the fluorescence intensity of a standard sample with the fluorescence intensity of an unknown sample to calculate the quantum yield of the unknown sample. Therefore, the obtained results depend on the accuracy of the standard sample’s quantum yield value.

In this application note, the fluorescence spectra for several samples will be obtained using an integrating sphere and the quantum yields will be calculated using the absolute method and compared with literature values.


Rhodamine B was used to correct the excitation spectrum prior to obtaining the absolute quantum yield calculations. The synchronous spectrum of a standard white diffuser plate was measured between 250 – 450 nm and the emission spectrum of a calibrated halogen light source was measured from 450 – 700 nm to correct the emission spectrum.

To measure the incident light using the integrating sphere, a spectrum is measured with nothing in the sample cell holder, as seen on the left in Figure 1. The sample is then placed into the integrating sphere and the spectrum is measured. The spectra of both the incident light and sample are shown in Figure 2. The incident photon (S0) peak appears in the excitation spectrum, illustrated in blue while the excitation and emission peaks indicate the number of photons unabsorbed (S1) and emitted (S2) by the sample, respectively.

Setup for measuring the incident light and (right) sample using the integrating sphere
Figure 1. (Left) Setup for measuring the incident light and (right) sample using the integrating sphere.
Excitation and emission spectra of incident (blue) and scattered (red) photons
Figure 2. Excitation and emission spectra of incident (blue) and scattered (red) photons.

Measurement Conditions

Quinine SulfateFluorescinTryptophan
Excitation Bandwidth5 nm5 nm5 nm
Emission Bandwidth5 nm5 nm5 nm
Excitation Wavelength350 nm475 nm280 nm
Scanning Speed200 nm/min200 nm/min200 nm/min
Data Interval0.5 nm0.5 nm0.5 nm
Response Time05. sec05. sec05. sec
PMT Voltage350 V250 V400 V

200 ppm of quinine sulfate in 1.0 N H2SO4, 15 ppm of fluorescein in 0.1 N aqueous NaOH, and 200 mg/mL of tryptophan in ultra pure water were prepared.


FP0008, FP-8500, ILF-835, FWQE-880, Integrating sphere, Fluorescence, Quantum yield, Absolute method,


The fluorescence spectra of quinine sulfate, fluorescein, and tryptophan are shown in Figures 3, 4, and 5, respectively.

Emission spectra of the incident light (blue) and quinine sulfate (red)
Figure 3. Emission spectra of the incident light (blue) and quinine sulfate (red).
Emission spectra of the incident light (blue) and fluorescein (red)
Figure 4. Emission spectra of the incident light (blue) and fluorescein (red).
Emission spectra of the incident light (blue) and tryptophan (red)
Figure 5. Emission spectra of the incident light (blue) and tryptophan (red).

Table 1 provides the values for the area under the maxima in the spectra shown in Figures 3-5.

Table 1. Peak areas from the fluoresce spectra of the three sample solutions.

Sample NameArea from Incident Light [S0 Area Scattered from Sample [S1Area Emitted from Sample [S2Scattered WL Range (nm)Emitted WL Range (nm)
Quinine Sulfate482672253814304320 - 365365 - 750
Fluorescein19174125156116465 - 485485 - 630
Tryptophan1361353584212101270 - 290290 - 550

The quantum yields for the three samples were calculated by the following equations:

The calculated quantum yield results using the values in Table 1 are shown in Table 2.

Table 2. Calculated quantum yield results.

Sample NameSample AbsorbanceInternal Quantum YieldExternal Quantum YieldInternal Quantum Yield Literature Values
Quinine Sulfate53.3%55.6%29.6%50 - 57%1
Fluorescein34.7%91.8%31.9%85 - 92%1
Tryptophan73.7%12.1%8.9%12 - 14%2


The quantum yields of quinine sulfate, fluorescein, and tryptophan have been calculated from the fluorescence spectra of the samples. The obtained results are within range of the published literature values.

Required Products and Software

  • FP-8300/8500/8600/8700 Spectrofluorometer
  • ILF-835 100 mm diam. Integrating Sphere
  • 1 mm liquid cell
  • FWQE-880 Quantum Yield Calculation program
  • ESC-842 Calibrated WI Light Source


1. The Spectroscopical Society of Japan, Japan Scientific Societies Press.
2. Principles of fluorescence spectroscopy, Joseph R. Lakowicz, Springer.

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About the Author

Leah Pandiscia received her PhD from Drexel University where she studied Biophysical Chemistry. She is a Spectroscopy Applications Scientist.