High performance capillary electrophoresis - T.E.A.M.

Instrumentation/Operation
4.3.7.2 Quantifying non-separated peaks
If two solutes are electrophoretically not separated, neither
qualitative nor quantitative analysis can be performed with
a single wavelength detector. With the diode-array, such
analysis can be performed even if the spectra overlap across
all wavelengths. This is accomplished through peak suppression
or signal subtraction using an extra wavelength, known
as the reference wavelength.
4.3.7.3 Validation of peak purity
It is important to determine whether electrophoretic peaks
are pure or if they consist of more than one solute. With
a diode-array detector, peak purity can be examined if the
spectra of the solute and the impurity are different. The
most common method involves acquiring several spectra
during the peak’s elution. By normalizing and overlaying the
spectra, the plots can be compared. When the spectra match
over all wavelengths, the peak can be considered pure.
Other validation methods include examining the absorbance
ratio at two wavelengths with the data plotted in the time
domain, spectral suppression, three-dimensional plotting
of the data, spectral deconvolution as a function of elution
time, and principal component analysis.
The peak purity function can be used when analysing non-
Gaussian shaped peaks. This is especially beneficial in CE
where peaks can be skewed as a result of conductivity
differences between the solutes and the running buffer.
Without this function, peak shape distortions can easily be
misconstrued as impurities. Significant effort in optimization
of buffer systems can be avoided by use of this spectral
information.
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