Molecular Devices SpectraMax ABS User Manual page 19

Note: You must select the PathCheck check box before a read because you cannot
apply the PathCheck technology after the read. After you read a plate with PathCheck
technology turned on, the software stores PathCheck information permanently within
the document.
Water Constant
The PathCheck technology is based on the absorbance of water in the near infrared spectral
region (between 900 nm and 1000 nm). If the sample is completely aqueous, has no turbidity
and has a low salt concentration (less than 0.5 M), the water constant correction method is
sufficient. The water constant is determined for each instrument during manufacture and is
stored in the instrument.
Note: The cuvette reference correction method that the software uses with the
PathCheck technology is different from the reference read of a cuvette that occurs
when you click the Ref button in the Cuvette Set section tool bar.
Eliminating the Pathlength Independent Component
Raw OD measurements of plate samples include both pathlength-dependent components
(sample and solvent) and a pathlength-independent component (OD of plate material). The
pathlength-independent component must be eliminated from the calculation to get valid
results that have been normalized by the PathCheck technology. You can do this using a plate
blank or using a plate background constant.
Using a Plate Blank
You can use this method if all samples in the plate are the same volume and the read does not
depend on the PathCheck technology to correct for variability in volumes.
To use a plate blank:
1. Designate a minimum of one well (preferably several) as Plate Blank.
2. Pipette buffer (for example, your sample matrix) into those wells and read along with the
samples. Do not use an empty well for a blank.
The instrument automatically subtracts the average of the blank wells from each of the
samples. The OD of the plate material is subtracted as part of the blank.
3. Select the Use Plate Blank check box in the Data Reduction dialog.
5065390 C
Chapter 4: Absorbance Read Mode
19