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Executing a Session
Pathlength correction
Pathlength correction
72
Thermo Scientific SkanIt
10. If you have a dispensing step in your protocol, you can empty the
dispenser tubing after measuring (see Emptying dispensers" on
page 76).
When a microplate is used to contain the samples, the beam traverses the
well from bottom to top. The pathlength is defined by the volume of liquid
that is added to each well. The longer the pathlength is, the greater the
absorbance will be. Therefore, the absorbance in a 1 cm cell (cuvette) will
be higher than that taken in a microwell. The pathlength correction factor
(K-Factor), when applied, accounts for these differences and allows for
samples measured in a microplate to be directly comparable to the same
samples measured in cuvettes.
The pathlength of a well can be measured using an absorbance measurement
of the liquid in each well at a wavelength where the solvent absorbs and the
photometric dye does not. The absorbance at such a wavelength is used to
correct the actual measured absorbance at the analytical wavelength for the
variation in well volume. All results of the correction calculation are
calculated for a 1 cm pathlength so that comparisons can be made to cuvette
data.
Photometric samples are commonly in a water-based buffer. Therefore, the
absorbance of water at a far infrared wavelength can be used for pathlength
correction. In SkanIt Software, the measurement is usually done with
wavelengths at 975 nm and 900 nm. The 975 nm measurement is used for
the calculation and the 900 nm measurement is used as a background value
for the 975 nm measurement. See Figure 5-1. This absorption band can be
used to calculate the pathlength of the aqueous solution being measured in
a microplate and then used to normalize the data.
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Software for Varioskan
User Manual
Thermo Fisher Scientific
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