Molecular Devices SpectraMax M2 User Manual page 16

When a Cuvette Reference is used for pathlength correction, the value of k is obtained by
taking optical density measurements on the fluid in the cuvette at two wavelengths, 1000
and 900 nm:
When the Water Constant is used for pathlength correction, the value of k is obtained
from the instrument. This constant is saved in the instrument in the factory and may
differ slightly from instrument to instrument.
Once the pathlength d is found, the following equation is used for the pathlength
correction:
PathCheck is applicable to almost all biological/pharmaceutical molecules in aqueous
solution because they have little or no absorbance between 900 nm and 1000 nm at
concentrations normally used. PathCheck can also be used with samples containing small
amounts of organics or high buffer concentrations by using the Cuvette Reference
(below).
2.4.1. WATER CONSTANT OR CUVETTE REFERENCE?
The PathCheck 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). When a non-interfering solvent is 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 SoftMax Pro, 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
calculations for the microplate samples.
SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D
(
k
= Cuvette OD OD
–
1000
OD
OD
Sample
-------- - -------------------------- -
=
( )
cm d cm
)
900
2.4. PathCheck
11