Agilent InfinityLab LC Series Diode Array Detectors User Manual
98
5
Optimizing the Detector
Optimizing the Detector Regarding to the System
absorbance from the reference bandwidth centered on the reference wavelength
will be subtracted from its equivalent value at the signal wavelength to produce
the output chromatogram.
The signal wavelength and bandwidth can be chosen so that they are optimized
for:
•
Broad band universal detection
•
Narrow band selective detection
•
Sensitivity for a specific analyte.
Broad band or universal detection works by having a wide bandwidth to detect
any species with absorbance in that range. For example, to detect all absorbing
molecules between 200 nm and 300 nm set a signal at 250 nm with a bandwidth
of 100 nm. The disadvantage is that sensitivity will not be optimal for any one of
those molecules. Narrow band or selective detection is used most often. The UV
spectrum for a particular molecule is examined and an appropriate absorbance
maximum is selected. If possible, the range where solvents absorb strongly
should be avoided (below 220 nm for methanol, below 210 nm for acetonitrile).
on page 99, anisic acid has a suitable absorbance
maximum at 252 nm. A narrow bandwidth of 4 nm to 12 nm generally gives good
sensitivity and is specific for absorbance in a narrow range.
The narrow band can be optimized for sensitivity for a specific molecule. As the
bandwidth is increased the signal is reduced but so is the noise and there will be
an optimum for best S/N. As an approximate guide, this optimum is often close
to the natural bandwidth at half-height of the absorption band in the UV
spectrum. In the anisic acid example this is 30 nm.
The analytical wavelength is usually set at a wavelength maximum to increase
sensitivity to that molecule. The detector is linear up to 2 AU and beyond for
many applications. This offers a wide linear range for concentration. For high
concentration analysis the concentration linear range can be extended by setting
the wavelength to one with a lower absorbance such as a wavelength minimum
or by taking a wider bandwidth which usually includes lower absorbance values.
The use of wavelength maxima and minima for quantitation dates back to
conventional UV detectors which because of mechanical tolerances in moving
gratings needed to avoid steeply sloping parts of the spectrum. Diode-array
based detectors do not have this limitation but for reasons of convention
maxima and minima are chosen in preference to other parts of the spectrum.
The reference bandwidth is normally set on a region of the UV spectrum in which
the analyte has no absorbance. This is shown in the spectrum for anisic acid in
on page 99. This spectrum is typical of many small molecules
containing a UV chromophore. For best results the reference has been set so that
