After connecting the column into the detector
it is time to prepare it for the analysis.
It is important to remember that the detector is the eyes of the
system. The baseline is actually the
detector's response to the mobile phase flowing out of the column
even before the injection and following it.
It can serve as an indication to the status of the column or the
solvents. If the column is polluted or
the solvents are not appropriate for HPLC work the baseline is not
stable. If the solvents are not
appropriate for HPLC
they can contain contaminations from the process of their production
that it is impossible to use them for gradients due to adsorption and
of the contaminations all the time while the composition of the mobile
The most abundant detector for HPLC is a
variable wavelength UV detector. The
wavelength is selected in advance and used throughout the injections.
- The selected wavelength should be appropriate for the
specific solutes used in the analysis. For
example, wavelength range for aromatic compounds with polar
substituents is 240-260 nm.
- If the compound does not have any absorption in the UV range
it is sometimes necessary to derivatize it with a UV-VIS absorbing
- Attempts to use wavelength range of 210-220 nm for the UV
detection can be very frustrating due to problems with absorption of
the mobile phase itself.
- Many UV-VIS detectors it is possible to use two wavelengths
in parallel, which can be helpful during development of a new method or
during adjustment of existing method to new compounds.
For example, one can try to work in two ranges
simultaneously, one wavelength selected from the range of 210-220 nm
and the other from the 240-260 nm simultaneously in order to detect as
many substances as possible in the sample.
- In a diode-array detector a full UV-VIS spectrum is
collected every acquisition point; therefore, it is frequently used for
method development and validation. The
optimal wavelength can be chosen, after screening all the spectra of
the peaks in the chromatogram.
For more information:
1-3: Solvent delivery system
4. The column
5 - Detector
(C) since 1997: Dr. Shulamit Levin