Absorbance refers to the ability of a substance to absorb light of a specific wavelength. In an microplate reader designed to measure absorbance, a light source illuminates the sample using a specific wavelength, selected by an optical filter or a monochromator, and a light detector located on the other side of the well measures how much of the initial light is transmitted through the sample: the amount of transmitted light will typically be related to the concentration of the molecule of interest, and the result is referred to as the Optical Density (OD) or absorbance of the sample. Absorbance is calculated as shown in the equation:
A λ = log10 (I0/I)
Where I is the intensity of light at a specified wavelength λ that has passed through a sample (transmitted light intensity) and I0 is the intensity of the light before it enters the sample.
In a typical colorimetric assay, an enzyme/substrate reaction
causes a color change in the samples in the microplate.
Light at a specific wavelength is passed through the sample to a detector.
The amount of light absorbed by the sample is referred to as the Optical Density (OD) or absorbance.
Many conventional colorimetric analyses have been, or can be miniaturized for measurement in a microplate reader. BioTek’s microplate readers and Gen5 software can facilitate assay miniaturization to microplates by providing optional cuvette ports and automated pathlength correction. Absorbance detection in microplate readers is used for assays such as ELISA assays, protein & nucleic acid quantification and purity assays, endotoxin analysis and many others.