Spectral Contrast Theory; Comparing Absorbance Spectra - Waters ACQUITY UPLC Operator's, Overview And Maintenance Manual

Spectral contrast theory

The spectral contrast algorithm compares the UV/Vis absorbance spectra of
samples the detector collects. This chapter describes the theory on which the
algorithm is based, explaining how it exploits differences in the shapes of the
absorbance spectra. It also explains how spectral contrast represents those
spectra as vectors, determining whether differences among them arise from
the presence of multiple compounds in the same peaks (coelution) or from
nonideal conditions such as noise, photometric error, or solvent effects.

Comparing absorbance spectra

When measured at specific solvent and pH conditions, the shape of a
compound's absorbance spectrum characterizes the compound. The varying
extent of UV/Vis absorbance occurring at different wavelengths produces a
unique spectral shape.
The following figure shows the absorbance spectra for two compounds, A and
B. The ratio of the absorbance at 245 nm to that at 257 nm is about 2.2 for
compound A and 0.7 for compound B. Note that this comparison of a single
wavelength pair's absorbance ratios yields little information about a
compound. For more information, you must compare the ratios of multiple
wavelength pairs.
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