1200 Series DAD and MWD User Manual 83
How to optimize the Detector 5
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 34 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 33 Wavelength Selection for Peak Suppression
WL1 (204 nm, caffeine)
WL2 (222 nm, hydrochlorothiazide
WL3 (260 nm, reference to suppress
hydrochlorothiazide
WL4 (282 nm, reference to suppress caffeine
Wavelength (nm)
WL2
WL3
WL4
WL1
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