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I am facing a ween problem. I am trying to find out, how would look like the graph for fluroscence of aesculine (intensity of fluoroscence on Y axis and pH on X axis).

I know, that increasing the pH causes a corresponding increase in the maximum fluorescence intensity from 77.74 (units) in the acidic sample to 146.13 at neutral pH and 232.69 at alkaline pH.

But, can I apply this on aesculine?

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  • $\begingroup$ You should measure the fluorescence vs that from a a standard compound excited at the same wavelength (with same absorbance) and observed at the same wavelength. If you look in the literature there are many standards and also compounds whose fluorescence yields are known. Look also for measuring relative fluorescence yields rather than absolute yields as these are v tricky. In any experiment you must ensure that you correct for any differences in the amount of light absorbed as this can be a source of large errors. $\endgroup$ – porphyrin Mar 22 at 16:22
  • $\begingroup$ A pH dependence is an indication that a pH buffer should be used in the sample matrix before the instrumental analysis. $\endgroup$ – MaxW Mar 22 at 17:11
  • $\begingroup$ I have no idea, you are talking about... $\endgroup$ – HASHTAG Mar 22 at 17:26
  • $\begingroup$ Keep also in mind that coumarins are labile at $\mathrm{pH} \ge 12$. $\endgroup$ – Mathew Mahindaratne Mar 22 at 17:50
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You should investigate several pH and study the effect of time on the fluorescence spectra. One cannot quote single values without mentioning the wavelengths of excitation and emission.

pH can alter the state of ionization and it can also cause hydrolysis of the sugar unit. You need to collect three different types of spectra at each pH, say from pH 2 to 12.

At each pH, first measure the absorption spectrum at those pH values. This is the easiest experiment. Do you see a difference in absorption spectra at different pH values? Note the trends. If yes, pH is affecting either the structure (decomposing) or the state of ionization.

Next, collect the excitation spectrum at a fixed emission wavelength (you have to estimate that). The excitation spectra should look similar to the absorption spectrum in shape.

Once you know the correct excitation wavelength at each pH, collect proper emission spectrum by using the lamba max of excitation.

So the answer is that be ready to see the changes in fluorescence spectra since there is no "the" fluorescence spectrum of a given molecule in a solution. You have to specify the solvent, pH, and excitation wavelength for experimental measurements.

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