Pathcheck Technology - Molecular Devices SpectraMax ABS User Manual

SpectraMax ABS and SpectraMax ABS Plus Microplate Spectrophotometers User Guide
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 Technology

The temperature-independent PathCheck technology normalizes 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 = εcL
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.
Horizontal
light path
PathCheck technology normalizes the data acquired from an absorbance read mode endpoint
read type to a 1 cm pathlength, correcting the OD for each well to the value expected if the
sample were read in a 1 cm cuvette. The instrument uses the factory installed water constant to
obtain the 1 cm values. For the SpectraMax ABS Plus you can read a cuvette that contains
deionized water or buffer to use the Cuvette Reference correction method (typically not
necessary when you use aqueous solutions with minimal alcohol, salt, or organic solvent
content).
18
Cuvette
Vertical light path
Microplate wells
5065390 C