Chapter 5: Read Modes and Read Types
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1. Fill a clean plate with water.
2. Read at the wavelengths you will use for the samples.
The average OD value is the Plate Background OD. If you intend to read your samples at
more than one wavelength, there should be a corresponding number of Plate Background
OD values for each wavelength.
Note:
It is important that you put water in the wells and do not read a dry plate for
the Plate Background OD. A dry plate has a slightly higher OD value than a water filled
plate because of differences in refractive indices. Use of a dry plate results in
PathCheck technology normalized values that are lower than 1 cm cuvette values.
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. Use of the Cuvette Reference does not reliably correct for turbidity.
Samples that are highly colored in the upper-visible spectrum might have absorbance that
extends into the near-infrared (NIR) spectrum and can interfere with the PathCheck
technology. Examples include Lowry assays, molybdate-based assays, and samples that
contain hemoglobins or porphyrins. In general, if the sample is distinctly red or purple, you
should check for interference before you use the PathCheck technology.
To determine possible color interference:
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 you
should 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 the solvent absorbs between
900 nm and 1000 nm, the interference would be similar to the interference of highly colored
samples. If you add 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.
