I am currently doing a fluorescence intensity scan with my compound. I am using the following parameters:
Excitation: 273-276nm
Slit: 20
Smoothing: 40
As my concentration increases from 20ug/ml -> 100ug/ml, the intensity decreases.
Could anyone explain this phenomenon? As I will need to do a standard curve.
Probably, it is the so-called "concentration quenching". Quenching is caused by the formation of non-fluoresent dimers due to increasing of concentration of your compound.
Try to work with more diluted solutuons.
This may happen when the concentration is so high that the excitation light is absorbed in the first millimeters of your sample. You can perhaps see this
or / and you can calculate the absorbance per mm of sample on the basis of the absorption coefficient of your compound for the low and high concentration
The usual practice is to set the excitation wavelength to the peak wavelength of the absorbance spectrum in order to maximize sensitivity of detecting fluorescence emission. However, if you are using high concentrations of the fluorescent compound, this may not be the best approach because it also maximizes the inner filter effect, which is self-absorption of light by the fluorophore that reduces the fluorescence intensity. One way to reduce this undesired effect is to shift the excitation wavelength off the peak absorbance wavelength. Another way is to use a cuvette with a smaller path length, such as a 4 mm square cuvette instead of 10 mm, or a 2 x 10 mm cuvette with the shorter dimension used for the excitation light..
The inner filter effect, and the methods to reduce it, can also apply to the emission wavelength if the compound has substantial absorbance at that wavelength.
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