Application Notes; Wavelength Selection - PerkinElmer 200 UV/VIS Series User Manual

Application Notes

Principles of Operation
Application Notes

Wavelength Selection

The proper selection of a wavelength for a particular analysis is paramount in the operation
of any absorbance detector. The choice of wavelength(s) can influence both the sensitivity
and selectivity of a method.
The first step in determining the optimal wavelength for a chromatographic method is to
obtain a UV absorbance spectrum for the compound(s) of interest. The UV spectrum should
be examined, and the wavelengths for absorbance maxima should be recorded. Spectra for
coeluting or interfering compounds should also be surveyed, and the UV cutoff for the mobile
phase should be noted. The ideal wavelength for an analysis will be that wavelength above
the UV cutoff for which an absorbance maximum exists which is fairly isolated from other
interfering peaks. A tradeoff may exist between choosing a difficult-to-use low wavelength
with high molar absorbtivity and an easy-to-use higher wavelength with low absorptivity.
It is not always necessary to choose the absolute absorbance maximum peak when choosing
a wavelength. If sensitivity is not an issue, a wavelength slightly above or below the absor-
bance maximum can be used to avoid an interfering peak. This technique of choosing a
wavelength off of the absorbance maximum peak can also be used to decrease the molar
absorptivity (sensitivity) for an analysis with concentrated solutes. This will help increase
the linearity of an analysis with high absorbance. However, if a wavelength on a steeply
sloped part of the UV spectrum is chosen, stability and linearity may be degraded.
There are many times when there are multiple compounds to be detected, each with a differ-
ent absorbance maxima. In these types of cases, there are two ways of dealing with wave-
length selection:
• Choose a general wavelength at which all compounds show some absorbance.
• Program a wavelength change using a timed events program.
After selecting a wavelength for the analysis, it is important that all analytical conditions
remain the same. Changes in temperature, pH, ionic strength, and the concentration of dis-
solved gases can all affect the absorbance or spectral profile of a compound. A change in the
absorbance profile of a compound will affect the sensitivity of the particular analysis. Any
changes in the composition or pH of the mobile phase should be kept to a minimum. Mobile
phases should be thoroughly degassed before use because dissolved oxygen and complexes
formed around oxygen can affect absorbance. Temperature shifts in the column eluent
should be avoided especially for analyses taking place at high sensitivities. Column and elu-
ent temperatures can be controlled through the use of a column oven.
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