Pathcheck Pathlength Measurement Technology And Interfering Substances - Molecular Devices SpectraMax M3 User Manual

0112-0115 F
SpectraMax M3, M4, M5, and M5
PathCheck Pathlength Measurement Technology
and Interfering Substances
Any material that absorbs in the 900 nm to 1000 nm spectral region
could interfere with PathCheck Pathlength Measurement Technology
measurements. Fortunately, there are few materials that do interfere at
the concentrations typically used.
Turbidity is the most common interference: if you can detect any
turbidity in your sample, you should not use the PathCheck Technology
feature. Turbidity elevates the 900 nm measurement more than the
1000 nm measurement and causes an erroneously low estimate of
pathlength. Using Cuvette Reference does not reliably correct for
turbidity.
Samples that are highly colored in the upper visible spectrum may have
absorbance extending into the near infrared (NIR) and can interfere
with the PathCheck Pathlength Measurement Technology. Examples
include Lowry assays, molybdate-based assays and samples containing
hemoglobins or porphyrins. In general, if the sample is distinctly red or
purple, you should check for interference before using the PathCheck
Pathlength Measurement Technology.
To determine possible color interference, do the following:
• Measure the optical density at 900 nm and 1000 nm (both
measured with air reference).
• Subtract the 900 nm value from the 1000 nm value.
• Do the same for pure water.
If the delta OD for the sample differs significantly from the delta OD for
water, then it is advisable not to use the PathCheck Technology feature.
Use of Cuvette Reference does not correct for the interference with the
current calculation scheme in the SoftMax Pro program. Currently,
Cuvette Reference involves a single (automated) read at 900 nm and
1000 nm and the automated calculations in the SoftMax Pro program
do not compensate for color or solvent interference. However, you
could correct for such interference by taking two cuvette
measurements and using a different set of calculations. For further
information, contact Molecular Devices Technical Support.
Organic solvents could interfere with the PathCheck Technology feature
if they have absorbance in the region of the NIR water peak. Solvents
such as ethanol and methanol do not absorb in the NIR region, so they
do not interfere, except for causing a decrease in the water absorbance
to the extent of their presence in the solution. Their passive
interference can be avoided by using the Cuvette Reference. If,
however, the solvent absorbs between 900 and 1000 nm, the
interference would be similar to the interference of highly colored
samples described above. If you are considering adding an organic
solvent other than ethanol or methanol, you are advised to run a
spectral scan between 900 nm and 1000 nm to determine if the solvent
would interfere with the PathCheck Technology feature.
Multi-Mode Microplate Readers
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