ThermoFisher Scientific NanoDrop Eight, User Manual

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Thermo Scientific NanoDrop Eight User Guide 121
Theory behind contaminant analysis
UV and UV-visible absorbance measurements are used to quantify nucleic acid and
protein samples at 260 nm and 280 nm, respectively. The analysis is based on the
fact that the total absorbance of a mixture solution at a given wavelength is the sum
of the absorbance values of each component in the mixture.
An ongoing challenge of this method is that a number of materials used in the
extraction process can absorb in various regions across the spectrum. When these
contaminants are present in a sample, they can interfere with the analysis by
artificially inflating the absorbance at the wavelength of interest, which causes the
analyte concentration to be overestimated.
Traditionally, purity ratios are used to detect the presence of contaminants that could
affect downstream applications. However, purity ratios do not always provide a
complete picture of possible contamination. When a purity ratio falls outside the
expected range, the spectral profile is often examined qualitatively.
Our Acclaro technology applies a quantitative approach to contaminant analysis.
Through sophisticated mathematical algorithms, Acclaro analyzes the spectral data
to identify probable contaminants in a sample and removes any contribution due to
the contaminant from the sample result. This results in a more accurate
concentration value of the analyte of interest and a more quantitative analysis of the
level of contamination.
Since the spectrum of a pure compound is unique to that compound, a mixture
spectrum of mostly known materials that have few interactions can be
mathematically broken down into its component spectra and the components
identified. The contaminant analysis algorithm uses a narrow spectral region
(220-285 nm) around the analysis wavelength (260 nm for nucleic acids, 280 nm for
proteins) to determine any absorbance contribution from possible known
contaminants (protein or nucleic acid, and phenol) that absorb in that region. The
entire spectrum is analyzed to determine the presence of other possible
contaminants such as guanidine HCl and/or guanidinium isothiocyanate, which are
common reagents used for nucleic acid purification.
Note Achieving consistent, high quality contaminant analysis results is
dependent on the quality of the measured sample spectra, which is dependent
on the maintenance status of the instrument. For more information, see
Maintenance Schedule .