5-8 Spectral Contrast Theory
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.
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. Keep in mind 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.
Solvent changes
As long as 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 figure “Effects of pH on the absorbance spectrum of p-aminobenzoic acid”
on page 5-9).
Threshold angle
In addition to computing spectral contrast angles, the spectral contrast
algorithm also computes a threshold angle. The threshold angle is the
maximum spectral contrast angle between spectra that can be attributed to
nonideal phenomena.
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