Molecular Devices SpectraMax Paradigm User Manual page 27

Interfering Substances
Material that absorbs in the 900 nm to 1000 nm spectral region could interfere with
PathCheck technology measurements. Fortunately, there are few materials that do interfere
at the concentrations generally used.
Turbidity is the most common interference. If you can detect turbidity in your sample, you
should not use the PathCheck technology. 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 might have absorbance
extending into the near-infrared (NIR) spectrum and can interfere with the PathCheck
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 technology.
To determine possible color interference, do the following:
Measure the OD 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
recommended to not use the PathCheck technology.
Organic solvents could interfere with the PathCheck technology 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. If, however, the solvent absorbs
between 900 nm and 1000 nm, the interference would be similar to the interference of highly
colored samples as previously described. If you are considering adding an organic solvent
other than ethanol or methanol, you should run a Spectrum scan between 900 nm and
1000 nm to determine if the solvent would interfere with the PathCheck technology.
5014038 E
Chapter 2: Read Modes and Read Types
27