Principles of Fluorescence Spectroscopy

52 INSTRUMENTATION FOR FLUORESCENCE SPECTROSCOPY
Figure 2.42. Comparison of the thermopile and the rhodamine B
quantum counter as radiation detectors. Redrawn from [26].
N H 2 SO 4 ) is useful at excitation wavelengths ranging from
220 to 340 nm and fluorescein (2 g/l in 0.1 N NaOH) is useful
over this same range, but is less reliable from 340 to 360
nm, 26 where absorption of fluorescein is weaker.
To record corrected excitation spectra, the quantum
counter is placed in the reference channel of the spectrofluorometer
(Figure 2.1). Because of the high optical density,
the reference cell holder is modified so that the emission is
observed from the same surface of the quantum counter that
is being illuminated. Alternatively, quantum counters can
be used in a transmission mode by observing the fluorescent
light exiting the back surface of the illuminated
cuvette. 27 In either case an optical filter is placed between
the quantum counter and the PMT, which eliminates incident
light but transmits the fluorescence. With a quantum
counter in place, a corrected excitation spectrum may be
obtained by scanning the excitation monochromator and
measuring the ratio of the fluorescence intensity from the
sample to that from the quantum counter. The wavelengthdependent
response of the emission monochromator and
phototube are not important because the emission wavelength
is unchanged during a scan of the excitation wavelength.
This procedure was used to record the corrected
excitation spectrum of fluorescein shown in Figure 2.6.
Other quantum counters have been described, and are
summarized in Table 2.3. The long wavelength dye HITC
extends the range to 800 nm, but its response is not as flat
as RhB. Unfortunately, there is no perfect quantum counter,
and for most applications RhB appears to be the best
choice.
Table 2.3. Quantum Counters
Solution Range (nm) Flatness Reference
3 g/l Rhodamine B in 220–580 "5% 26
ethylene glycol
8 g/l Rhodamine B in 250–600 "4% 27
ethylene glycol a
2 g/l Fluorescein in 240–400 b "5% 26
0.1 N NaOH
4 g/l quinine sulfate 220–340 "5% 26
in 1 N H 2 SO 4
Rhodamine in polyvinyl 360–600 "3% 28 c
alcohol (PVA) films
Coumarins in PVA films 360–480 "3% 28 c
5 g/l Ru(bpy) 3 2+ in 360–540 1.1% 29
methanol
Ru(bpy) 3 + in PVA films 360–530 1% 29 d
8 g/l HITC e in acetonitrile 320–800 f "10% 30
aA higher concentration of RhB is claimed to be preferred for use in
transmission mode. See [27].
bResponse may be 15% lower from 340 to 360 nm.
cSee [28] for details on the rhodamines, coumarins, and PVA film
preparations.
dSee [29] for details.
eHITC, 1,1',3,3,3',3'-hexamethylindotricarbocyanine.
fDeviation up to 20% occurs near 470 nm.
2.9. CORRECTED EMISSION SPECTRA
2.9.1. Comparison with Known Emission Spectra
It is necessary to know the wavelength-dependent efficiency
of the detection system to calculate the corrected emission
spectra. It is difficult and time consuming to measure
the correction factors for any given spectrofluorometer.
Even after careful corrections are made the results are only
accurate to "10%. For this reason the observed technical
spectra are usually reported. If corrected spectra are necessary,
one simple and reliable method of obtaining the necessary
correction factors is to compare the observed emission
spectrum of a standard substance with the known corrected
spectrum for this same substance. Such spectra have
been published for a variety of readily available fluorophores
including quinine sulfate, β-naphthol, 3-aminophthalimide,
4-dimethylamino-4'-nitrostilbene, and N,Ndimethylamino-m-nitrobenzene.
31–36 The emission wavelengths
of these compounds cover the range from 300 to
800 nm and the data are presented in graphical and numerical
form. Corrected spectra have been published for a
series of harmine derivatives, covering the range 400–600