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The fluorescence excitation spectra show the change in fluorescence intensity as a function of the wavelength of the excitation light.

I'm interested in the certain physical implications of the above.

Does that mean, that at the wavelength of excitation maximum the most of the molecules get excited? Or do they just emit more photons per molecule per unit time?

Additionally, if the former is correct, is there any relationship between the percentage of excited molecules and the excitation spectra?

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The fluorescence excitation spectra show the change in fluorescence intensity as a function of the wavelength of the excitation light. Does that mean, that at the wavelength of excitation maximum the most of the molecules get excited? Or do they just emit more photons per molecule per unit time?

You have to understand how the excitation spectrum is collected. You have to know (or at least have an estimate) of the emission maximum of a given molecule. For example, if we have quinine, I should know that it emits blue light and maximum wavelength of emission is ~ 450 nm.

Now we will fix the emission wavelength at 450 nm and scan the entire UV-VIS range for excitation. Say, we would excite quinine with 200 to 700 nm. One wavelength at a time, and count how many photons corresponding to 450 nm were emitted. This plot is called the excitation spectrum.

An excitation spectrum basically tells you which wavelength (from a given instrument) is going to show the highest intensity. I emphasized "a given instrument" because an excitation spectrum has to be corrected for the light source intensity. It will vary from instrument to instrument. Nobody corrects it because it is a long procedure as unfortunately, normalization cannot correct it.

Anyway, a corrected excitation spectrum should match in shape with the absorption spectrum provided that there is only one fluorophore in the molecule.

I wanted to connect the excitation spectrum with an absorption spectrum because we can now connect the molar absorptivity with the excitation. Consider molar absorptivity as a measure of probability of excitation. Higher the value of molar absorptivity, more molecules will absorb the light and hence more will emit. So your first interpretation is correct. It is a collective behavior of the all the molecules which are being illuminated in the cuvet.

Also remember that excitation emission is normally a one photon excitation and one photon emission in majority of cases until and unless you are using fancy lasers. So you cannot have one photon in, and multiple photons out in routine cases. In rarer cases, you can indeed have two photon in for excitation and one photon out. You need lasers again.

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Good books have been written to explain all the implications of fluorescence spectroscopy. There is no universal answer because of the complications of the experiment. In general there are atomic spectra and molecular spectra.

  • Atomic spectra tend to give line spectra.
  • For molecules however the binding energy of the electron depends not only on the electronic energy but also the vibrational state of the molecule. This coupling creates a microstate and thus molecules tend to give broad peaks.

There is also the Stokes Shift which indicates that the exciting photon must be of greater energy than the emitted photon. This is mostly true, but there is also resonance radiation, and anti-Stokes radiation.

Given:

The fluorescence excitation spectra show the change in fluorescence intensity as a function of the wavelength of the excitation light.

Does that mean, that at the wavelength of excitation maximum the most of the molecules get excited?

Maybe...

Normally when trying to measure excitation sensitivity you'd normalize the number of excitation photons. Think of it as how many emission photons would be given off per excitation photon versus the energy of the exciting photon. (This will normally be considered to be less than 1). However a real excitation spectra need not have the same intensity of photons at each energy. So a lot of excitation photons at a non-optimal energy might product more excitation photons than the optimal energy at a lower intensity.

Or do they just emit more photons per molecule per unit time?

In general one excitation photon creates one excited state which may decay by the emission of a photon.

There are of course lasers where the atoms or molecules are pumped into an excited state then one photon can stimulate the multiple emissions.

Additionally, if the former is correct, is there any relationship between the percentage of excited molecules and the excitation spectra?

The fluorescent quantum yield is the number of excitation photons emitted per excited state.

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