Molecular Devices SpectraMax M2 User Manual page 25

SpectraMax M2 and M2e Microplate Reader User Guide
The factor of two comes from the fact that %T is expressed as a percent of the transmitted
light and log
When in %Transmittance analysis mode, the instrument converts the raw OD values
reported by the instrument to %Transmittance using the above formula. All subsequent
calculations are done on the converted numbers.
Applications of Absorbance
Absorbance-based detection is commonly used to evaluate changes in color or turbidity,
permitting widespread use including ELISAs, protein quantitation, endotoxin assays, and
cytotoxicity assays.
Optimizing Absorbance Read Mode
You can adjust the wavelength of the transmitted light in 1-nm increments between 200 nm
and 1000 nm. You can also use the instrument for reading up to six wavelengths per plate,
which allows for reference wavelength readings such as A260 and A280 for nucleic
determination.
For an assay blank, you should use appropriate plate blanks or group blanks in a template
that you define in the software. You can also use the PathCheck Pathlength Measurement
Technology feature to normalize the data to a 1 cm pathlength.
PathCheck Pathlength Measurement Technology
The temperature-independent PathCheck® Pathlength Measurement Technology
normalizes your absorbance values to a 1 cm path length based on the near-infrared
absorbance of water.
The Beer–Lambert law states that absorbance is proportional to the distance that light
travels through the sample:
A = εcL
where A is the absorbance, ε is the molar absorptivity of the sample, c is the concentration of
the sample, and L is the pathlength. 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 do extinction-based
assays and makes it confusing to compare results between microplate readers and
spectrophotometers.
The standard pathlength of a 1 cm cuvette is the conventional basis to quantify the unique
absorptivity properties of compounds in solution. Quantitative analysis can be done on the
basis of extinction coefficients, without standard curves (for example, NADH-based enzyme
assays). When you use a cuvette, the pathlength is known and is independent of sample
volume, so absorbance is directly proportional to concentration when there is no
background interference.
25
(100) = 2.
10
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