Waters 2996, Operator'S Manual

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Comparing Absorbance Spectra 41
5
Chapter 5
Spectral Contrast Theory
This chapter explains the theory behind the Spectral Contrast technique, which is used to
compare UV/Vis absorbance spectra collected by the 2996 Detector. Spectral Contrast
makes use of the fact that different compounds have differently shaped absorbance
spectra. This chapter describes how Spectral Contrast represents absorbance spectra as
vectors. When applied to the UV/Vis absorbance data collected by the 2996 Detector, the
Spectral Contrast technique determines whether differences between spectra are due to the
presence of multiple compounds in the same peaks (coelution) or due to nonideal
conditions such as noise, photometric error, or solvent effects.
5.1 Comparing Absorbance Spectra
The shape of an absorbance spectrum is determined by the relative absorbance at different
wavelengths. The shape of a compound’s absorbance spectrum is a characteristic of that
compound at the solvent and pH conditions under which the absorbance spectrum is
measured.
Figure 5-1 shows the absorbance spectra for the two compounds, A and B. The ratio of the
absorbance at 245 nm to the absorbance at 257 nm is approximately 2.2 for compound A
and 0.7 for compound B.
The absorbance ratios of two wavelength pairs is a limited spectral comparison. For more
information, you need to compare the absorbance ratios of multiple wavelength pairs.