Fluorolog-3 v. 2.2 (11 Jul 2002)
Applications
10-6
Because triplet transitions are “forbidden” quantum-mechanically, the average phos-
phorescence decay times are generally longer, ranging from a few microseconds to sev-
eral seconds. Thus, phosphorescence offers a longer observation period for monitoring
reactions, looking at environmental effects on a sample, or following changes in the
hydrodynamic characteristics of macromolecular systems.
In phosphorescence experiments using the Fluorolog
®
-3 and the FL-1042 Phos-
phorimeter Assembly, the sample is excited by a pulsed light source. Acquisition of the
emission signal is synchronized to the pulse, with user-specified delay and sampling
times, to produce time-resolved spectral data. With an appropriate choice of delay time,
the user may select only the luminescence of interest.
Time-resolved data-acquisition also makes it possible to acquire phosphorescence de-
cay curves and compute lifetimes of lanthanides such as europium and terbium, as well
as the biological probe eosin.
Low-temperature scans
One way to protect a sample from molecular collisions that can quench luminescence is
by isolating the sample in a rigid matrix. Thus, cooling with liquid nitrogen enhances
the phenomenon of fluorescence, even for seemingly dormant samples. In addition, the
superior resolution of a Fluorolog
®
-3 double-grating spectrometer system optimizes
measurements under these conditions.
Monitoring kinetic reactions using time-
based fluorescence
By setting the wavelengths at the excitation and emission peaks of a sample, the
Fluorolog
®
-3 systems can monitor fluorescence as a function of time. This permits the
use of Fluorolog
®
-3 systems in reaction-rate determinations, which monitor the forma-
tion or breakdown of a fluorescing species. Reaction-rate determinations are highly se-
lective. Because only
changes
in intensity are considered, the method is not affected by
interference from continuous background signals or steady-state scatter.
Front-face detection to enhance data
collection for absorbent or solid samples
Fluorescence typically is collected at right angles (90°) from transmitted or scattered
light. Yet right-angle viewing is inappropriate for some samples. Imprinted paper, for
example, reflects light, which interferes with accurate data collection. In highly absor-
bent samples like hemoglobin or milk, most of the emitted light is reabsorbed before
the fluorescence can be measured. A significant design feature of the Fluorolog
®
-3 Sin-
gle-Beam and T-Box spectrofluorometers is that they offer a choice between conven-
tional right-angle or front-face fluorescence detection. Front-face viewing is ideal for
solid, turbid or highly absorbent samples such as pellets, powders, and monolayers on
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