Absorbance Read Mode - Molecular Devices SpectraMax Paradigm User Manual

Absorbance Read Mode

In the Absorbance (ABS) read mode, the instrument measures 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
where I
through the sample, and A is the measured absorbance.
For Absorbance reads, you can choose whether to display absorbance data as Optical
Density (OD) or %Transmittance (%T) in the Reduction dialog.
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 optimum results, Molecular Devices recommends that you run replicates for all blanks,
controls, and samples. In this case, the blank value that can 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
%T = 100T
where I is the intensity of light after it passes through the sample and I
before it enters the sample.
Optical Density and %Transmittance are related by the following formulas:
%T = 10
OD = 2 – log
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 SoftMax Pro Software converts the raw OD
values reported by the instrument to %Transmittance using the above formula. All
subsequent calculations are done on the converted numbers.
5014038 E
(I /I) = –log (I/I )
10 0 10 0
is incident light before it enters the sample, I is the intensity of light after it passes
0
0
2–OD
(%T)
10
(100) = 2.
10
Chapter 2: Read Modes and Read Types
is incident light
0
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