Molecular Devices SpectraMax M2 User Manual page 15

2. Principles of Operation
SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D
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based assays and also makes it confusing to compare results between microplate readers
and spectrophotometers.
The standard pathlength of a cuvette is the conventional basis for quantifying the unique
absorbtivity properties of compounds in solution. Quantitative analyses can be performed
on the basis of extinction coefficients, without standard curves (e.g. NADH-based enzyme
assays). When using a cuvette, the pathlength is known and is independent of sample
volume, so absorbance is proportional to concentration.
In a microplate, 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 arise from pipetting the samples and standards. The
PathCheck feature automatically determines the pathlength of aqueous samples in the
microplate and normalizes the absorbance in each well to a pathlength of 1 cm. This
novel approach to correcting the microwell absorbance values is accurate to within 2.5%
of the values obtained directly in a 1-cm cuvette.
Horizontal
light path
Figure 2.1: Cuvette and microwell light paths.
Reference measurements made by reading the cuvette (Cuvette Reference) or using
factory-stored values derived from deionized water (Water Constant) can be used to
normalize the optical density data for microplate wells.
PathCheck pathlength correction is accomplished only when using the SoftMax Pro
software. PathCheck is patented by Molecular Devices and can be performed only on an
MDC plate reader.
The SpectraMax M2 and SpectraMax M2
Water Constant methods.
The actual pathlength, d, of a solvent is found from the following equation:
Sample OD
( )
d cm -------------------------------------------------------------------- -
=
Vertical light path
Cuvette Microplate wells
e
offer both the Cuvette Reference and the
( )
OD
–
1000 900
k