Consider a substance is used at with an concentration x, which is having absorbance greater than 4, how to troubleshoot the problem where x is a critical concentration.
Hello Sambit Mandi
Microplate readers will show overflow if the well absorbance is more than 4.0 OD because of the presence of high concentration of the substance under investigation. 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. Measurements from 2.5 - 4.0 OD use 0.3%- 0.01% of the original light and therefore these measurements have lower resolution and accuracy.
Therefore, 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 true for all microplate readers as well as spectrophotometers.
You can solve your problem by diluting your substance, and reading the absorbance of the diluted sample. You can then multiply the result by the dilution factor.
For example, if you dilute your substance by 10 fold, the concentration of the substance will be 10 times less. So you will record an absorbance reading that may be below 4.0 OD. Try to dilute your substance in such a way that the aborbance reading for the substance falls between 0.1 and 1.0 OD because this range gives the highest accuracy. You may then multiply the result by the dilution factor to obtain the final concentration of the undiluted substance.
If you have diluted your substance 100 times to give an OD value of 0.5 (say), then you multiply the concentration of your substance, say (y) which you obtain for this absorbance with the dilution factor. In this case the dilution factor is 100.
So, 100 x concentration of the diluted substance(y)= x (concentration of undiluted substance.
Best.
In microplate readers, absorbance greater than 4 can lead to overflow because most microplate readers have a limited dynamic range of detection. This means that the maximum absorbance that can be accurately measured is limited, and values beyond this range will appear as "overflow" or "out of range" on the instrument.
To troubleshoot this problem, there are a few options:
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