Agilent Technologies 1200 Infinity Series User Manual page 103

5
Optimizing the Detector
Optimizing the Detector Regarding to the System
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 Figure 29 on page 104. This spectrum is typical of many small
molecules containing a UV chromophore. For best results the reference has
been set so that it is a wide band as close to the signal wavelength as possible
but on a zero absorbance region. Reference bandwidths of 60 nm to 100 nm
are commonly used. The default reference is 360 nm with a bandwidth of
100 nm. A wide bandwidth is used because this reduces the noise in the
reference signal (from statistical theory, the error, i.e. noise in this case, is
reduced by the square root of the number of determinations). It is important
that the reference bandwidth does not extend to a part of the spectrum that
has some absorbance as this would then reduce the resulting signal and
sensitivity would be reduced. The use of a reference wavelength can help to
reduce drift or wander in the chromatogram caused by refractive index
changes due to room temperature fluctuation or gradient operation. The effect
of a reference signal can be easily tested by setting two otherwise identical
signals, one with and one without a reference signal. If there is no part of the
spectrum with zero absorbance then it will be better to have the reference
signal turned off.
Agilent 1200 Infinity Series DAD User Manual 103