Chapter 5: Read Modes and Read Types
5090339 A
29
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.
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.
Using a Plate Background OD
If your sample volumes are not identical or if you choose not to use a Plate Blank, then you must
use a Plate Background OD. Omitting a Plate Background OD results in artificially high values
after being normalized by the PathCheck technology.
To determine the Plate Background OD:
1. Fill a clean plate with water.
2. Read at the wavelengths you will use for the samples.
The average OD value is the Plate Background OD. If you intend to read your samples at more
than one wavelength, there should be a corresponding number of Plate Background OD values
for each wavelength.
Note: It is important that you put water in the wells and do not read a dry plate for the
Plate Background OD. A dry plate has a slightly higher OD value than a water filled plate
because of differences in refractive indices. Use of a dry plate results in PathCheck
technology normalized values that are lower than 1 cm cuvette values.
