Water Constant Or Cuvette Reference; Background Considerations - Molecular Devices SpectraMax M3 User Manual

Principles of Operation
22

Water Constant or Cuvette Reference?

The PathCheck Pathlength Measurement is based on the absorbance of
water in the near infrared 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 is adequate. The
Water Constant is determined during manufacture and is stored in the
instrument.
If the sample contains an organic solvent such as ethanol or methanol,
we recommend using the cuvette reference. It is important that the
solvent does not absorb in the 900 nm to 1000 nm range (to determine
whether or not a given solvent would interfere, see the discussion of
interfering substances below).
added to the aqueous sample, the water absorbance decreases
proportionally to the percentage of organic solvent present. For
example, 5% ethanol decreases the water absorbance by 5% and
results in a 5% underestimation of the pathlength. You can avoid the
error by putting the same water/solvent mixture in a cuvette and using
the Cuvette Reference.
To use the Cuvette Reference, place into the cuvette port a standard
1 cm cuvette containing the aqueous/solvent mixture that is used for
the samples in the microplate. The cuvette must be in place when you
read the microplate. When you click the Read button in the SoftMax Pro
program, the instrument first makes the 900 nm and 1000 nm
measurements in the cuvette, and then makes the designated
measurements in the microplate. The cuvette values are stored
temporarily and used in the PathCheck Pathlength Measurement
Technology calculations for the microplate samples.
Use of Cuvette Reference with PathCheck Pathlength Measurement
Technology is different from a reference reading of a cuvette in a
CuvetteSet section (by clicking the Ref button in the CuvetteSet section
tool bar in the SoftMax Pro program). The cuvette reference used for
PathCheck Pathlength Measurement Technology calculations
(measurements at 900 nm and 1000 nm) does not produce data that
can be viewed in a CuvetteSet section and is used only with data in
microplates, not cuvettes.

Background Considerations

Raw optical density measurements of microplate samples include both
pathlength-dependent components (sample and solvent) and a
pathlength-independent component (OD of microplate material). The
latter must be eliminated from the PathCheck Pathlength Measurement
Technology calculation in order to obtain PathCheck Technology-
normalized results. There are 3 ways to accomplish this: plate blanks,
plate background constants, and plate pre-reads, all of which are
described in the PathCheck Pathlength Measurement Technology
section of the SoftMax Pro User Guide.
When a non-interference solvent is
0112-0115 F