Optimizing Selectivity; Quantifying Coeluting Peaks By Peak Suppression - Agilent Technologies InfinityLab LC Series User Manual

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
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
Agilent InfinityLab LC Series Diode Array Detectors User Manual
WL1 (204 nm, caffeine)
WL2 (222 nm, hydrochlorothiazide)
WL3 (260 nm, reference to suppress)
hydrochlorothiazide)
WL4 (282 nm, reference to suppress caffeine)
WL3
Wavelength (nm)
WL1 WL2
Wavelength Selection for Peak Suppression
WL4
Figure 28 on
94