Correct microplate absorbance readings to a standard one centimetre pathlength.
Microplate readers measure vertically through the liquid, so the pathlength depends on fill volume rather than being fixed at one centimetre as in a cuvette. Correcting for it is what makes plate readings comparable with cuvette values. A 200 microlitre well in a standard 96-well plate has a pathlength near 0.5 cm, so uncorrected readings understate concentration by roughly half.
Spectrophotometer Pathlength Correction
Beer-Lambert: A = ε·c·l, so correcting to 1 cm means dividing by the actual pathlength
Beer-Lambert: A = ε·c·l, so correcting to 1 cm means dividing by the actual pathlength Microplate readers measure vertically through the liquid, so the pathlength depends on fill volume rather than being fixed at one centimetre as in a cuvette. Correcting for it is what makes plate readings comparable with cuvette values.
A 200 microlitre well in a standard 96-well plate has a pathlength near 0.5 cm, so uncorrected readings understate concentration by roughly half.
This calculator takes 4 inputs: Measured absorbance, Well volume, Well diameter, Molar extinction coefficient. The pre-filled defaults are a realistic starting point — replace them with figures from your own environment for a result you can act on.