Chapter 5: Read Modes and Read Types
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where I is the intensity of light after it passes through the sample and I
0
is incident light
before it enters the sample.
Optical Density and %Transmittance are related by the following formulas:
%T=10
2–OD
OD=2–log
10
(%T)
The factor of two comes from the fact that %T is expressed as a percent of the transmitted
light and log
10
(100)=2.
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. 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.
You can use the Protocol Libraries to quickly find and open a predefined protocol.
You can download additional protocols and updated protocols from the Protocol Home
Page button on the Protocols tab. (www.softmaxpro.org).
PathCheck Pathlength Measurement Technology
The temperature-independent PathCheck® Pathlength Measurement Technology
normalizes your absorbance values to a 1cm 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 - b = c d e + f
where a is absorbance, b is blank, c is concentration, d is the depth of sample layer, e is
extinction (coefficient of...), and f is further terms, e.g., non-linearity caused from turbidity.
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.
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