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When I measure optical density in a microplate reader the absorbance is proportional to the concentration and the height of the liquid. Is Beer-Lambert law also valid for fluorescence and luminescence?

Let's say I prepare a fluorescent solution and measure e.g. the signal of 100 μL and of 50 μL. Do I get the half signal for 50 μL?

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No the Beer Lambert law ($I_{trans}=I_0\exp(-\epsilon[C]L)$) does not apply as this shows that the transmitted light intensity decreases exponentially with concentration as it passes through a solution.

Fluorescence/luminescence is directly proportional to the concentration of molecules excited provided that the solution is dilute so that dimers or excimers are not formed and that self-absorption of the fluorescence light by another part of the same solution does not occur.

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  • $\begingroup$ Thanks for your answer. So beside the Beer's Law when I would measure 3 different volumes e.g. [25, 50, 75 uL] with the same fluorescence concentration will I receive a linearity, as more volume means more light or is that wrong? The reason why I am asking is about volume verification for pipetting devices. Optical density could do that too but the dynamic range isn't very high $\endgroup$ – cekar Jan 21 at 14:18
  • $\begingroup$ Yes if you account for all porphyrin said. But even so you should integrate all the emitted light . Not viable at all. I would rather trick in a way that you always measures the same sample and reasons on dilution steps. This let you know if you are really picking up 25 50 75 ... $\endgroup$ – Alchimista Jan 21 at 15:42

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