Principles Of Operation; Absorbance; Optical Density; Transmittance - Molecular Devices SpectraMax M2 User Manual

2.1.

ABSORBANCE

Absorbance is the amount of light absorbed by a solution. To measure absorbance
accurately, it is necessary to eliminate light scatter. In the absence of turbidity, absorbance

= optical density.

where I is incident light, and I is transmitted light.
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In this user guide, we use the terms absorbance and optical density interchangeably.
2.2. OPTICAL DENSITY
Optical density is the amount of light passing through a sample to a detector relative to
the total amount of light available. Optical density includes absorbance of the sample plus
light scatter from turbidity.
2.3.

TRANSMITTANCE

Transmittance is the ratio of transmitted light to the incident light.
where I is incident light, and I is transmitted light.
0
2.4.

PATHCHECK

The Beer-Lambert law states that absorbance is proportional to the distance that light
travels through the sample:
where A is the absorbance,
and c is the concentration of the sample. In short, the longer the pathlength, the higher
the absorbance.
Microplate readers use a vertical light path so the distance of the light through the sample
depends on the volume. This variable pathlength makes it difficult to perform extinction-
SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D

2. Principles of Operation

⁄
( )
A
= log I I
0
( I ⁄ )
T = I
0
%T
= 100T
εbc
A
=
ε
is the molar absorbtivity of the sample, b is the pathlength
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