HPLC HINTS & TIPS for
Chromatographers
Tip# 109: UV / VIS
DETECTOR SIGNAL BANDWIDTH:
Modern UV/VIS detectors offer the operator a choice of one to
several hundred different signal wavelength choices (as is the case for Diode Array
Detectors). Besides being able to specify a single wavelength, you often can choose a
signal bandwidth (bw) to associate with each wavelength [e.g. for a 280 nm signal
with 10 nm bandwidth this is often written as: 280 (10) or 280:10]. Signal Bandwidth is
the total number of nanometers across the specific signal value chosen. For example: If
you selected a signal wavelength at 280 nm and chose a bandwidth value of 10 nm, then you
are actually gathering all signal data from 275 nm to 285 nm (5 nm to the left of the apex
and 5 nm to the right for a total of 10 nm). Using a narrow bandwidth has the advantage of
increasing the signal selectivity of the detector as you are only collecting data within a
tight window. If you were to increase the bandwidth to 60 nm in the same example you would
now be collecting data starting at 250 nm and continuing through to 310 nm. The additional
data collected over this wide range can reduce the total noise (by averaging it over a
wide range), improve the S/N ratio (increases sensitivity), but it also reduces the
selectivity. Large bandwidths also increase the chance you may include peak signal data
from other co-eluting components into your signal data. You must select a bandwidth range
which is always 'safe' from any other potential peak. As with many things in life there is
a balance between selectivity and sensitivity.
When developing methods we recommend that you initially choose a
bandwidth value of 10 nm for each signal. This provides a nice balance between selectivity
and sensitivity. It is also a common bandwidth value used on detectors which have a fixed
signal bandwidth (such as many single or variable wavelength detectors).
If you have determined the exact signal maximum for your sample and
you would like to gain additional sensitivity for your sample (and thus decrease
selectivity), re-run the analysis using several different increasing signal bandwidth
values (e.g. 10, 20, 30, 50 and 100 nm). Choose bw values that are safely within the range
of the detector and also away from any potential co-eluting peaks. *Be sure and calculate
the actual signal to noise ratio of the peak of interest after each analysis. This is a
critical step! Do not be fooled by increases in the peak height or area alone as these
changes are not always synonymous with better signal to noise ratios. Only by measuring
the actual baseline noise level for each run and comparing it with the actual peak signal
obtained will you be able to determine if increasing the bandwidth has provided you with
better noise reduction and signal strength.
To increase spectral signal selectivity choose a bw value that is
very narrow. A value such as 2 or 4 nm would allow the detector to collect only signal
data that is at or near the apex of your selected wavelength. This can be very useful when
trying to discriminate your signal from nearby signal peaks,
especially at low wavelengths such as 210nm.
> Bill Letter, 12/06/10.
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