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Fluorescence Spectroscopy open open
  • Fluorescence Spectroscopy
  • Principles and Theory of Fluorescence Spectroscopy
  • What is the Jablonski Diagram?
  • What is a Fluorescence Measurement?
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  • What is Fluorescence Anisotropy or Fluorescence Polarization?
  • What are Luminescence Quantum Yields?
  • What is Ratiometric Fluorescence?
  • What is an Excitation Emission Matrix (EEM)?
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  • What is Singlet Oxygen?
  • How to Calculate Signal to Noise Ratio
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    What are Luminescence Quantum Yields?

What are Luminescence Quantum Yields?

An integrating sphere fiber-coupled to a fluorometer for PLQY measurements

Fig. 18: An integrating sphere fiber-coupled to a fluorometer for PLQY measurements.

The Photoluminescence quantum yield or PLQY of a molecule or material is defined as the number of photons emitted as a fraction of the number of photons absorbed. This characteristic property of a fluorophore or fluorescent molecule is important for understanding molecular behavior and interactions for many key materials.

Similarly, the electroluminescence quantum yield, or ELQY, is the number of photons emitted divided by the electron current of a device. This is important for lighting, display devices, and photovoltaic materials.

Electroluminescence can be measured using an integrating sphere (left) by fitting a powered device such as an LED into the sample tray

Fig. 19: Electroluminescence can be measured using an integrating sphere (left) by fitting a powered device such as an LED into the sample tray. Center: The integrated intensity can be measured with input voltage or current. Right: Color can be plotted in CIE 1931 coordinates by measurement of the spectrum in the sphere.

The materials for which PLQY and ELQY are used are:

  • Photovoltaics and Solar cells
  • Novel nanomaterials
    • Nanoparticles
    • Quantum dots
    • Graphene/ Single Walled Carbon nanotubes
  • Lighting and display materials (LEDs, OLEDs)
  • Coordination chemistry
  • Films, coatings
  • Electrovoltaics
  • Cured/doped polymers, gels, hydrogels
  • Paints, coatings, colorimetry

There are three ways to measure PLQY: the comparison method, fluorescence lifetime, and the direct method (integrating sphere).

How do I use the comparative method to determine quantum yield?

Usage the comparative method to determine quantum yield.

Fig. 20: Left: The equation for calculating the fluorescence quantum yield of an unknown (QF) by comparing it to the spectrum of a known standard. Right: A table of some known PLQY standards and their respective excitation wavelengths and quantum yields.

In the Comparative method, one uses a reference standard, a sample with known emission and absorbance properties close to that of the sample of interest, and has a known PLQY value. The absorbance and fluorescence of the reference standard are measured and then the same is measured for the sample under study.

The following equation is used where QF is the quantum yield of the unknown fluorescent sample, QR is the quantum yield of the reference standard, IF and IR are the integrated fluorescence intensities for the unknown and the reference, respectively, and AF and AR are the absorbance values of the unknown and reference, respectively. A limited amount of reference standards make this method somewhat limited as well.

How do I use fluorescence lifetimes for quantum yield determination?

Usage of fluorescence lifetimes for quantum yield determination.

Fig. 21: Fluorescence Quantum yield equation calculated by the rate constants of fluorescence (kf), non-radiative dissipation (knr) and energy transfer (kt). Fluorescence lifetime calculated by one over the sum of the rate constants. And the quantum yield in relation to the Stern-Volmer quenching constant (K), the biomolecular quenching constant (kq) and the lifetime ( t0). (Lakowicz, 2006)

There is a method that uses fluorescence lifetimes and different concentrations of a quencher to calculate the quantum yield of a molecule.

The equation on the right is used where tf is the quantum yield, and kf, knr, and kt are the rate constants of fluorescence, non-radiative dissipation and energy transfer, respectively, τf is the fluorescence lifetime of the sample. PLQY is determined by the rate constants of these non-radiative processes that compete with fluorescence such as FRET and Stern-Volmer quenching.

Fluorescence excitation and emission spectra for different concentrations of sodium ascorbate quenching fluorescence from 9-aminoacridine.

Fig. 22: Left: Fluorescence excitation and emission spectra for different concentrations of sodium ascorbate quenching fluorescence from 9-aminoacridine. Right: Lifetime and intensity ratios (I/I0 and t/t0) versus concentrations. Linear fits to these plots yield quenching constants.

By adding a dilution series of quencher to a fluorescent solution, the PLQY can be calculated by finding the Stern- Volmer quenching constants (K) and the bimolecular quenching constant (kq). While this is certainly a robust method, it requires a good amount of sample preparation and is not convenient for solid samples.

How do I use an integrating sphere for quantum yield measurements?

The integrating sphere method of measuring PLQY.

Fig. 23: Left: An integrating sphere where samples are placed on the inside of the sphere and then fluorescence is measured. Right: The reflectance spectrum of Spectralon® material that coats the inside of an integrating sphere. (LabSphere Spectralon(R) datasheet, 2017)

The integrating sphere method is a direct method of measuring PLQY. A sphere is coated with an all reflective surface, such as barium sulfate-based materials or Spectralon® to capture all the light going in and out of the sphere.

A measurement is done of the fluorescence emission (Ec) and the scatter (Lc) of the sample and also the emission and scatter of a blank (La and Ea). From these two spectral measurements (sample and blank), the PLQY can be calculated from the equation in Fig. 24.

Quantum yield equation from measurement using an integration sphere.

Fig. 24: Quantum yield (Ff) equation from measurement using an integration sphere.

Where Eb is the integrated luminescence from the sample caused by indirect luminescence from the sphere and A is the absorbance of the sample at the excitation wavelength. A simple calculator that incorporates the two traces along with appropriate spectral correction factors is used to give the PLQY and associated error analysis.

Calculation of PLQY from scatter (left) and fluorescence (right) of blank and sample

Fig. 25: Calculation of PLQY from scatter (left) and fluorescence (right) of blank and sample

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