Technical data

62 Agilent 1290 Infinity LC System Manual and Quick Reference
3 Optimization of the Agilent 1290 Infinity LC System
How to Achieve Higher Sensitivity
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 23 on page 62. 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.
Figure 23 Spectrum of Anisic Acid