Waters 2998, Overview And Maintenance Manual

Page: 65 / 94

December 5, 2017, 715004753 Rev. B
Page 65
5.3.2 Differences between spectra of the same compound
Small but significant differences between absorbance spectra can result from factors other than
the absorbance properties of different compounds. For example, multiple spectra of the
same
compound may exhibit slight differences because of detector noise, photometric error, high sample
concentration, or variations in solvent conditions. The spectra in the next figure, for example, show
how instrument noise can affect the shape of an absorbance spectrum of one compound at two
concentrations, one high and one low. Note that the spectral contrast angle between these
absorbance spectra of the same compound is 3.4

.
Figure 5–5: Normalized absorbance spectra of a compound at two concentrations
5.4 Undesirable effects
Shape differences between absorbance spectra can be caused by one or more of the following
undesirable effects:
• Detector noise
• Photometric error caused by high sample concentration
• Variation in solvent composition
These sources of spectral variation can cause chemically pure, baseline-resolved peaks to exhibit
a small level of spectral inhomogeneity. You can assess the significance of spectral inhomogeneity
by comparing a spectral contrast angle to a threshold angle (see
page 67 ).
Normalized spectra of a compound at different
concentrations
Spectral contrast angle: 3.4

Wavelength (nm)
Region of little or no
analyte absorption
N
ormalized absor
bance