SpectraMax Mini User Guide
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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 the solvents have
absorbance in the region of the NIR water peak. Solvents such as ethanol and methanol do not
absorb in the NIR region, so the solvents do not interfere, except to cause 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.
Fluorescence Intensity Read Mode
Fluorescence occurs when absorbed light is re-radiated at a longer wavelength. In the
Fluorescence Intensity read mode, the instrument measures the intensity of the re-radiated light
and expresses the result in Relative Fluorescence Units (RFU).
The governing equation for fluorescence is:
Fluorescence = extinction coefficient × concentration × quantum yield × excitation intensity
× pathlength × emission collection efficiency
Fluorescent materials absorb light energy of a characteristic wavelength (excitation), undergo
an electronic state change, and instantaneously emit light of a longer wavelength (emission).
Most common fluorescent materials have well-characterized excitation and emission spectra.
The following figure shows an example of excitation and emission spectra for a fluorophore.
The excitation and emission bands are each fairly broad with half-bandwidths of approximately
40 nm, and the difference between the wavelengths of the excitation and emission maxima (the
Stokes shift) is generally fairly small, about 30 nm. There is considerable overlap between the
excitation and emission spectra (gray area) when a small Stokes shift is present.
