Agilent Technologies 1200 Infinity Series User Manual page 97

5
Optimizing the Detector
Optimizing Selectivity
With a UV-visible detector based on a diode array and the correct choice of a
reference wavelength setting, quantitative detection is possible. To suppress
caffeine, the reference wavelength must be set to 282 nm. At this wavelength,
caffeine shows exactly the same absorbance as at 222 nm. When the
absorbance values are subtracted from each another, any indication of the
presence of caffeine is eliminated. In the same way, hydrochlorothiazide can
be suppressed if caffeine is to be quantified. In this case the wavelength is set
to 204 nm and the reference wavelength to 260 nm. Figure 26 on page 97
shows the chromatographic results of the peak suppression technique.
The trade-off for this procedure is a loss in sensitivity. The sample signal
decreases by the absorbance at the reference wavelength relative to the signal
wavelength. Sensitivity may be decreased by as much as 10–30 %.
Figure 26 Peak Suppression Using Reference Wavelength
Agilent 1200 Infinity Series DAD User Manual 97