Frequently Asked Questions

Microplate Instrumentation

Question

Why are performance specifications of microplate readers limited to 2.5 OD when the reader can measure samples which have absorbance values up to 4.0 OD?

Answer

Spectrophotometers including microplate readers do not perform with high accuracy and precision at OD greater than 2.5 OD. The reason for this is that the amount of light that is transmitted through the sample is very, very small. See the formula below:

A = OD = -log T
or
A = OD = log (1/T)

Thus, measurements from 2.5 -4.0 OD use than 0.3%- 0.01% of the original light and therefore these measurements have lower resolution and accuracy. For this reason, we do not quote performance statistics for our readers above 2.5 OD.

Measurements of samples greater than 2.5 OD should not be used to obtain quantitative measurements. For the highest accuracy and precision, only use measurements with absorbance values between 0.10 and 1.0 OD which is equivalent to 10% to 90% of light transmission.

This is a fundamental quality of spectrophotometric technology and will be true of all microplate readers as well as spectrophotometers.


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