# Is Beer-Lambert law also valid for fluorescence and luminescence?

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?

## 1 Answer

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.

• 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 – cekar Jan 21 at 14:18
• 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 ... – Alchimista Jan 21 at 15:42