VersaMax, SpectraMax 340PC384, 190, Plus 384 Microplate Readers User Guide
20
0112-0126 B
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.
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 =
ε
cL
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.
In a plate, pathlength is dependent on the liquid volume, so absorbance is proportional to
both the concentration and the pathlength of the sample. Standard curves are often used to
determine analyte concentrations in vertical-beam photometry of unknowns, yet errors can
still occur from pipetting the samples and standards. The PathCheck technology determines
the pathlength of aqueous samples in the plate and normalizes the absorbance in each well
to a pathlength of 1 cm. This way of correcting the microwell absorbance values is accurate
to within ±4% of the values obtained directly in a 1 cm cuvette.
