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Chapter 5: Read Modes and Read Types
Absorbance Read Mode
The instrument uses the Absorbance (ABS) read mode to measure the Optical Density (OD) of
the sample solutions.
Absorbance is the quantity of light absorbed by a solution. To measure absorbance accurately,
it is necessary to eliminate light scatter. If there is no turbidity, then absorbance = optical
density.
A = log
10
(I
0
/I) = –log
10
(I/I
0
)
where
I
0
is intensity of the incident light before it enters the sample divided by the light after it
passes through the sample, and
A
is the measured absorbance.
The instrument allows you to choose whether to display absorbance data as Optical Density
(OD) or %Transmittance (%T).
Optical Density
Optical density (OD) is the quantity of light passing through a sample to a detector relative to
the total quantity of light available. Optical Density includes absorbance of the sample plus light
scatter from turbidity and background. You can compensate for background using blanks.
A blank well contains everything used with the sample wells except the chromophore and
sample-specific compounds. Do not use an empty well for a blank.
Some applications are designed for turbid samples, such as algae or other micro-organisms in
suspension. The reported OD values for turbid samples are likely to be different when read by
different instruments.
For optimal results, you should run replicates for all blanks, controls, and samples. In this case,
the blank value that will be subtracted is the average value of all blanks.
% Transmittance
%Transmittance is the ratio of transmitted light to the incident light for absorbance reads.
T = I/I
0
%T = 100T
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.
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