Agilent InfinityLab LC Series Diode Array Detectors User Manual 95
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 29 on page 95 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 29 Peak Suppression Using Reference Wavelength
Time (min) Time (min)
Wavelength 204 nm
No reference
Hydrochlorothiazide
and caffeine
Wavelength 222 nm
No reference
Hydrochlorothiazide
and caffeine
Hydrochlorothiazides
uppressed
Wavelength 204 nm
Reference 260 nm
Caffeine suppressed
Wavelength 222 nm
Reference 282 nm
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