Molecular Devices SpectraMax Paradigm User Manual page 24

SpectraMax Paradigm Multi-Mode Detection Platform User Guide
Applications of Absorbance
Absorbance-based detection has been commonly used to evaluate changes in color or
turbidity, permitting widespread use including ELISAs, protein quantitation, endotoxin
assays, and cytotoxicity assays. With absorbance readers that are capable of measuring in
the ultraviolet (UV) range, the concentration of nucleic acids (DNA and RNA) can be found
using their molar extinction coefficients.
For micro-volume measurements, you can use SpectraDrop 24-well Micro-Volume
Microplates and SpectraDrop 64-well Micro-Volume Microplates.
To do absorbance reads, the SpectraMax Paradigm Multi-Mode Detection Platform requires
the Absorbance Detection Cartridge, see page
You can use the Protocol Manager in the SoftMax Pro Software to quickly find and open a
predefined protocol.
More protocols and updated protocols can be downloaded from the Knowledge Base on the
Molecular Devices support web site (www.moleculardevices.com/support) or from the
protocol sharing web site (www.softmaxpro.org).
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 = εbc
where A is the absorbance, ε is the molar absorptivity of the sample, b is the pathlength, and
c is the concentration of the sample. 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 also makes it confusing to compare results between microplate readers and
spectrophotometers.
The standard pathlength of a 1 cm cuvette is the conventional basis for quantifying 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 using 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.
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