Measuring fluorescence 1-5
Scanning
Detectors equipped with excitation and emission monochromators can easily
scan a range of excitation or emission wavelengths. Changing the wavelength
involves changing the monochromator setting. During a scan, the setting on
one monochromator is held constant while the other monochromator scans a
range of wavelengths.
Multichannel operation
Detectors equipped with excitation and emission monochromators can change
the wavelength of the excitation and emission settings. In multichannel
operation, both monochromators move rapidly between the selected
wavelength pairs to produce multiple chromatogram traces. Multiple outputs
can then derive additional information from a single separation.
Fluorescence data
Detectors report their data in units of fluorescence intensity (emission) or
energy. In addition, the 2475 detector reports intensity using normalized
units to compensate for variability between individual detectors and offset any
age-related decrease in lamp intensity. When using normalized units, changes
in gain improve the signal-to-noise ratio but do not change the peak response,
conferring a high degree of bench-to-bench reproducibility of fluorescent
signal measurements.
Emission units and normalization
The 2475 detector offers two types of output units: emission and energy.
Emission units are normalized to a standard water reference, and their
magnitude is as independent of the PMT gain as possible. You can compensate
for changes that normally influence the signal strength of fluorescence
measurements, such as lamp or optics degradations, by periodically
renormalizing to the standard water reference. Renormalizing reduces
variations in fluorescence signal strengths from one detector to another.
The following equation calculates the emission units value (EU) at any time
(t):
EU
t
= (PMTCounts
t
/ Gain
t
) × (Gain
Raman
/ Counts
Raman
) × 100
where
To Next Page
Loading...
