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Homework Help: Question on fluorescence

  1. Oct 29, 2008 #1

    I have a quick question on fluorescence. I have fluorescein molecule. Its absorbance is pH sensitive. However, I want to know if

    1. It emits fluorescence.

    2. the fluorescence intensity pH sensitive

    I know for sure it emits fluorescence. I see, what I believe to be an emission peak. However, I am not able to get any pH response. Irrespective of how acidic or basic I get, I am unable to get the intensity to change.
    Can anyone explain this. Can we say by default that if a molecule has fluorescein in its structure, it should fluoresce? And, if it fluoresces, then it should be pH sensitive too, given, the absorbance is pH sensitive.

    Thanks for your help.
  2. jcsd
  3. Oct 30, 2008 #2


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    Could you be more specific? What molecule is it and how are you adjusting the pH?
  4. Nov 2, 2008 #3
    The molecule is fluorescein (sounds like a typo, but it is a commonly used fluorescent dye). Whether or not the absorption or emission peak intensity changes with pH depends on what wavelength/peak you are looking at. Fluorescein has an isosbestic point where the intensity of the peak at that point is independent of pH (other peaks still depend on pH). The reason why the isosbestic point is independent of pH is that both the conjugate base and conjugate acid have the same extinction coefficent at this point. It doesn't matter how much of one or the other you have at the isosbestic point (amounts determined by the pH), you will get an absorbance based on the total concentration only.
  5. Nov 4, 2008 #4
    Thank you for the replies. When I say this molecules is fluorescein, I mean it has fluorescein in its structure. Its absorbance is a pH sensitive. This molecule does have an isobestic point for absorbance. In that case, would it mean it should have an isobestic point for fluorescence too? Only that I haven't come across any literature stating its fluorescent properties. I see an absolutely steady fluorescence for neat 0.01M NaOH and 0.01M HCl solutions. Absolutely no change whatsoever.
    However, I am not sure I fully grasp the idea behind isobestic point. I did not come across this for any of the fluorophores I have worked with previously. Can you please tell me more about it...or guide me on where I can find more information on this.

    Thank you for your help.
  6. Nov 5, 2008 #5


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    Does the molecule have more than one acid / base site?
  7. Nov 11, 2008 #6


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    What was the concentration of your solution? If the concentration is too high, you will see no change in apparent fluorescence. This is due to the strong absorption of the fluorophore. Did the solution appear to fluoresce from only the surface of the solution or did it appear that the entire cuvette glowed?

    Beers law (and nonlinearities for Beers law) works for both absorbance and emission.

    To either confirm or eliminate this as a possible cause, you need to look at the absorbance of serial dilutions of the tested solution at both the absorbance frequencies (one each for the protonated form and the deprotonated form) and the absorbance at the emission frequencies. The Beers law plot of the serial dilutions should be linear for all cases... if not you are observing the nonlinearity of the absorption/emission and misinterpreting it as the chormophore having no pH dependence.
    Last edited: Nov 11, 2008
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