Waters 2998, Overview And Maintenance Manual

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December 5, 2017, 715004753 Rev. B
Page 66
5.4.1 Detector noise
Statistical and thermal variations add electronic noise to the detector’s absorbance
measurements. The noise, which manifests itself as fluctuations in the baseline, is known as
baseline noise. The magnitude of any absorbance differences caused by statistical and thermal
variations can be predicted from the instrument noise in the baseline region of a chromatogram.
5.4.2 Photometric error
At high absorbances (generally those greater than 1 AU), a combination of effects can produce
slight departures (about 1%) from Beer’s law because of photometric error. Although photometric
errors at this level can negligibly affect quantitation, they can nevertheless be a significant source
of spectral inhomogeneity. To minimize the effects of photometric error for all spectral contrast
operations, the maximum spectral absorbance of a compound should be less than 1 AU. Note that
the absorbance of the mobile phase reduces the working linear dynamic range by the amount of
mobile phase absorbance at each wavelength. For examples of mobile phase absorbance, see
Appendix C .
See also:
For more information about the effects of the photometric error curve, refer to
Principles
of Instrumental Analysis
, 3rd. ed., by Douglas A. Skoog, Saunders College Publishing, 1985, pp.
168–172.
5.4.3 Solvent changes
If solvent concentration and composition do not change (isocratic operation), background
absorbance, if any, by the solvent remains constant. However, change in solvent pH or
composition, such as that which occurs in gradient operation, can affect the intrinsic spectral
shape of a compound (see the following figure).
Figure 5–6: Effects of pH on the absorbance spectrum of p-aminobenzoic acid
TP02837
Effect of pH
pH 6.9
Wavelength (nm)
Absorba
nc
e
pH 5.1
pH 3.1