Is an emission spectrum really independent of excitation wavelength?

[dentedduck]

I've often read that the emission spectrum of a fluorescent molecule is independent of the wavelength used for the excitation. But what happens in the case of a small Stoke's shift where the excitation and emission wavelengths overlap?

If I use a narrow band excitation with a wavelength in the overlap region then the energy of the excitation light would be lower than the highest energy photons in the emission. Wouldn't that break conservation of energy? I would expect the bandwidth of the emission to be limited in that case.


Dave

 

[Ygggdrasil]

Even though the emitted photons have more energy than the absorbed photons, this does not break the conservation of energy. Rather, what actually occurs is a phenomenon called "fluorescent cooling." In essence, the laser picks off the highest energy particles from the ground state population, and relaxation from the excited state puts them (on average) into a lower energy level of the ground state.

See, for example, Epstein et al. 1995 Observation of laser-induced fluorescent cooling of a solid. Nature: 377 500. doi:10.1038/377500a0
(free version: http://usna.edu/Users/physics/mungan/Publications/Pub-Nature.php)

 

[Yinxiao Li]

Even though the emitted photons have more energy than the absorbed photons, this does not break the conservation of energy. Rather, what actually occurs is a phenomenon called "fluorescent cooling." In essence, the laser picks off the highest energy particles from the ground state population, and relaxation from the excited state puts them (on average) into a lower energy level of the ground state.

See, for example, Epstein et al. 1995 Observation of laser-induced fluorescent cooling of a solid. Nature: 377 500. doi:10.1038/377500a0
(free version: http://usna.edu/Users/physics/mungan/Publications/Pub-Nature.php)

You are an expert on this! But I have a question. Since photobleaching might happen to fluorophore, then the emission spectrum might change? Today I did my experiment and I found that under an excitation wavelength of 532nm and 785nm, the emission spectra are different! Moreover, the emission spectrum from 785nm does not look like a good spectrum...Any clue on what is happening? Thank you so much!

 

[Ygggdrasil]

It's hard to say what's going on w/o knowing more about the experiment. What molecule are you looking at? Does the molecule show significant absorbance at 785 nm in its absorption spectrum? These two wavelengths are very far apart such that they probably represent distinct transitions and would not be expected to show similar emission spectra.

 

[Yinxiao Li]

It's hard to say what's going on w/o knowing more about the experiment. What molecule are you looking at? Does the molecule show significant absorbance at 785 nm in its absorption spectrum? These two wavelengths are very far apart such that they probably represent distinct transitions and would not be expected to show similar emission spectra.

The molecule that I am looking at is MPS-PPV (one kind of conjugated polyelectrolyte, bought from Sigma Aldrich). The absorption spectrum shows a peak at ~451nm and decreases to very small absorbance after ~600nm. So there is very little absorbance in 785nm, according to the absorbance spectrum. However, even under 785nm, I can still get something. Should I trust this result? Or maybe I will neglect 785nm and go with 532nm?

 

[Ygggdrasil]

I'd worry that it is noise, especially since you say that it does not look like a typical emission spectrum. I will note that since you are measuring a solution containing a mixture of polymers of different lengths, the emission spectrum can vary with the excitation wavelength as different wavelengths may excite different populations of molecules (as opposed to the case where all of the fluorophores in solution are identical where the overall shape of the emission spectrum does not typically depend on the excitation wavelength).

 

[Yinxiao Li]

I'd worry that it is noise, especially since you say that it does not look like a typical emission spectrum. I will note that since you are measuring a solution containing a mixture of polymers of different lengths, the emission spectrum can vary with the excitation wavelength as different wavelengths may excite different populations of molecules (as opposed to the case where all of the fluorophores in solution are identical where the overall shape of the emission spectrum does not typically depend on the excitation wavelength).

As you said, "since I am measuring a solution containing a mixture of polymer of different lengths, the emission spectrum can vary with excitation wavelength". I have an extending question that can the emission spectrum change from trial to trial under the same excitation wavelength? In fact, I have measured the MPS-PPV in solution under confinement for several times and the results are not exactly the same. I attached the files. Is it normal to have such a variation?

Another question is that most people measure emission spectrum with the molecule of interest in solution (I also did this), can I measure it after the molecule is dried? I have tried it with MPS-PPV, and the spectra are different!

 

[Ygggdrasil]

As you said, "since I am measuring a solution containing a mixture of polymer of different lengths, the emission spectrum can vary with excitation wavelength". I have an extending question that can the emission spectrum change from trial to trial under the same excitation wavelength? In fact, I have measured the MPS-PPV in solution under confinement for several times and the results are not exactly the same. I attached the files. Is it normal to have such a variation?

Some of the differences could be due simply to measurement error, but I'm not familiar enough with the spectroscopy of conjugated polymers to say whether the variability you see is normal.

Another question is that most people measure emission spectrum with the molecule of interest in solution (I also did this), can I measure it after the molecule is dried? I have tried it with MPS-PPV, and the spectra are different!

The spectra of fluorescent molecules often depends strongly on their environment (e.g. many fluorophores are pH deptendent, and things like solvent polarity and viscocity can alter their photophysical properties). So, I would absolutely expect the spectra of the dried molecule to differ from the spectra when your polymer is in solution.