Agilent InfinityLab LC Series Diode Array Detectors User Manual 94
5 Optimizing the Detector
Optimizing Selectivity
Optimizing Selectivity
Quantifying Coeluting Peaks by Peak Suppression
In chromatography, two compounds may often elute together. A conventional
dual-signal detector can only detect and quantify both compounds independently
from each other if their spectra do not overlap. However, in most cases this is
highly unlikely.
With a dual-channel detector based on diode-array technology, quantifying two
compounds is possible even when both compounds absorb over the whole
wavelength range. The procedure is called peak suppression or signal
subtraction. As an example, the analysis of hydrochlorothiazide in the presence
of caffeine is described. If hydrochlorothiazide is analyzed in biological samples,
there is always a risk that caffeine is present which might interfere
chromatographically with hydrochlorothiazide. As the spectra in Figure 28 on
page 94 shows, hydrochlorothiazide is best detected at 222 nm, where caffeine
also shows significant absorbance. It would therefore be impossible, with a
conventional variable wavelength detector, to detect hydrochlorothiazide
quantitatively when caffeine is present.
Figure 28 Wavelength Selection for Peak Suppression
WL3
WL4
WL2
WL1
Wavelength (nm)
hydrochlorothiazide)
WL1 (204 nm, caffeine)
WL4 (282 nm, reference to suppress caffeine)
WL3 (260 nm, reference to suppress)
WL2 (222 nm, hydrochlorothiazide)
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