Fluorescence Spectrum

Fluorescence spectra are much more highly structured than absorbance spectra,
often exhibiting several distinct maxima of fluorescence intensity with respect to
both excitation and emission wavelengths.

From: Treatise on Geochemistry (Second Edition), 2014

Related terms:

Photon, Protein, Ion, Fluorescence Intensity, Absorption Spectrum, Wavelength,

Fluorescence, Emission Spectrum, Laser Induced Fluorescence Spectroscopy

View all Topics

Rare-earth-doped chalcogenide glass

for lasers and amplifiers
J. Heo, W.J. Chung, in Chalcogenide Glasses, 2014

11.2.2 Pr3 +- and Dy3 +-doped chalcogenide glasses for the 1.3 μm

(O-band) fiber amplifiers
Fluorescence spectra in Fig. 11.1 recorded from the glass doped with 0.1% Dy3 +
show the interesting intersity changes of two fluorescence changes at 1.31 μm
(6 F11/2 · 6H9/2 → 6H15/2) and 1.75 μm (6H11/2 → 6H15/2) (Shin et al., 2001). In Ge-Ga-S
glass, the intensity of the 1.31 μm emission is considerably smaller than that
centered at 1.75 μm; similar to the spectrum reported previously (Wei et al., 1994).
On the other hand, the 1.31 μm emission intensity clearly increased at the expense
of the 1.75 μm fluorescence intensity in glasses containing bromides (KBr and CsBr).
The similar phenomenon on the relative intensities of these two emissions was also
observed in glasses containing iodides.
 11.1. Emission spectra of the 1.31 μm (6 F11/2, 6H9/2 → 6H15/2) and 1.75 μm (6H11/2 →-
 6H15/2) transitions in Dy3 +-doped glasses (Shin et al., 2001).

Addition of CsBr into Ge-Ga-S glasses did not always result in the enhancement of
the lifetimes and quantum efficiencies of the excited energy levels. For instance,
Fig. 11.2(a) shows emission spectra for three Dy3 +-doped glasses in Ge-As-Ga-S with
different amounts of CsBr. Emission spectra from glasses containing 4% and 5%
CsBr were similar to those of Ge-Ga-S glasses (Wei et al., 1994) with a weak 1.31 μm
emission and a relatively strong 1.75 μm emission. On the other hand, the glass with
> 5% of CsBr showed a widely different fluorescence spectrum and became similar
to that of the 10% CsBr-containing glass shown in Fig. 11.1.
11.2. (a) Emission spectra and (b) decay curves of Dy3 +:6 F11/2 · 6H9/2 → 6H15/2 (1.31 μm)
transition in (0.95-y)Ge0.25As0.1S0.65-0.05GaS3/2-yCsBr glasses.

One of the major drawbacks for Dy3 +-doped sulfide glasses has been the short
lifetime (~ 38 μs, shown in Fig. 11.2(b)) of the upper emission level (6 F11/2 · 6H9/2)
of the 1.31 μm fluorescence. This is due to the high multiphonon relaxation rate (~
25,000 sec–1) of the 6 F11/2 · 6H9/2 → 6H11/2 transition in sulfide glasses. When the
appropriate amount of CsBr was added, the lifetime of the 6 F11/2 · 6H9/2 level with
0.1 at.% Dy3 + doping was 1320 μs, and it is approximately 35 times longer than that
measured from normal sulfide glasses (Shin et al., 2002). Quantum efficiency ( )
was less than 20% but glasses containing CsBr provided the quantum efficiency
approaching 100%. Decay curves also showed notable changes with CsBr addition
(Shin et al., 2002).
> Read full chapter

Fluorophores: Descriptions and Prop-

erties
J.R. Albani, in Structure and Dynamics of Macromolecules: Absorption and Fluores-
cence Studies, 2004

3b Effect of the viscosity on the fluorescence emission spec-

trum
Fluorescence spectra are also dependent on the rigidity of the medium. Relaxation
phenomenon (reorientation of the dipole environment) occurs much easily when the
medium is fluid. In such case, emission will occur after relaxation. This is the case
when relaxation is faster than fluorescence, i.e. the relaxation lifetime r is shorter
than the fluorescence lifetime 0. This occurs when the binding site is flexible and
the fluorophore can move easily. Displacements of fluorophore molecules will be
representative of the time scale and amplitude of the motions of the surrounding
protein matrix. Emission from a relaxed state does not change with the excitation
wavelength. This can be explained by the fact that whatever the value of the excitation
wavelength, emission will occur always from the same energy level.

When the binding site is rigid, fluorescence emission occurs before relaxation. In this
case, excitation at the longer wavelength edge of the absorption band photoselects
population of fluorophores energetically different from that photoselected when the
excitation wavelength is shorter.

When the red edge excitation is performed, the energy hvedge of the electronic
transition is equal to Eeedge-Eeedge, where Ee – Eg refers to the difference of energy
between excited and ground states; hvedge is lower than hvsw, the energy of the
electronic transition that occurs at short wavelengths. Thus excitation at the red edge
gives a fluorescence spectrum with a maximum located at higher wavelength than
that obtained when excitation is performed at short wavelengths.

Thus, the observation of red shift of em,max upon red shift in ex, indicates that the
system meets with the o < r condition. This means a decreased mobility of the
fluorophore on its binding site with respect to the dipolar matrix of the site.

In other words, when the surrounding environment of the fluorophore or when

its binding site is rigid, the emission maximum of the fluorophore will shift to
higher wavelengths with the excitation wavelength. In general, emission maxima are
compared if the spectra are symmetric. Otherwise, the centers of gravity should be
compared. The red-edge excitation spectra method can be applied to Trp residues
(Fig. 3.41) and to extrinsic fluorophores such as TNS, calcofluor or any other fluo-
rophores.

Figure 3.41. Red-edge excitation of holo-Phel 10Ser azurin. Dependence of the

fluorescence spectrum peak of holo-Phel10Ser as a function of the excitation wave-
length, at 1 bar (filled symbols) or at 2000 bar (open symbols). At 1 bar, the shift of
the emission peak is larger than that at 2000 bar. The decrease in the spectral shift in
presence of hydrostatic pressure suggests a higher relaxation of the Trp48 microen-
vironment, due to the destabilization of the protein structure.Source: Mei, G., Di
Venere, A., Campeggi, F. M., Gilardi, G., Rosato, N., De Matteis, F. and Finazzi-Agrò,
A. 1999, European Journal of Biochemistry 265, 619-626. Authorization of reprint
accorded from Blackwell Publishing.

Finally, we should indicate that emission from a low polar or apolar environment
yields a fluorescence emission spectrum located at low wavelength such as when
the environment is rigid. Also, a highly dynamic environment yields a fluorescence
emission spectrum with a maximum located at long wavelengths such as when the
polarity of the environment is high. Therefore, one should be careful in interpreting
the results when work is performed on macromolecules.

An example to illustrate this. Ascorbate oxidase is a copper-containing enzyme

which catalyzes a redox between vitamin C and molecular oxygen. The protein is
a homodimer of monomers, each containing three domains and 14 tryptophan
residues.

X-ray diffraction studies (Messerschmidt et al. 1989; 1992) have shown that six tryp-
tophan residues / monomer are completely shielded from the solvent, six others are
buried but not completely and two are exposed to the solvent. Figure 3.42 displays
the position of the emission peak of protein emission with excitation wavelength, in
absence and presence of 1.3 M CsCl.

Figure 3.42. Red-edge fluorescence emission of ascorbate oxidase. Maximum fluo-

rescence emission versus excitation wavelength for Ascorbate oxidase (circles) and
for ascorbate oxidase in presence of 1.3 M CsCl (squares). Solid lines represent the
best linear fits of circles.Source: Di Venere, A., Mei, G., Gilardi, G., Rosato, N., De
Matteis, F., McKay, R., Gratton, E. and Finazzi Agro, A. 1998. Eur. J. Biochem. 257,
337-343. Authorization of reprint accorded from Blackwell Publishing.

We notice that up to 295 nm, the peak moves slowly to longer emission wavelength,
above 297 nm this dependence becomes steeper and within 10 nm (297 −307 nm)
the spectrum shifts to the red by about 11 nm. The authors correlated this effect to
a significant different exposure of the classes of tryptophan residues to the solvent.
In fact, when the experiment was carried out in the presence of CsCl, two slopes
were still visible but the difference was much less pronounced. This experiment by
itself shows that the protein contains tryptophan residues that are exposed to the
solvent.

Addition of cesium quenches the fluorescence of these tryptophans and the main
fluorescence will occur mainly from buried Trp residues. In the present data, we do
not have any direct proof of the dynamic aspect. Although one should expect that
the tryptophan residues that are exposed to the solvent would rotate much faster
than the other tryptophan residues. The authors showed this result by measuring
the anisotropy as a function of wavelength in the absence and in the presence of
1.3 M CsCl (Di Venere et al. 1998).

> Read full chapter

Six-membered Rings with Two Het-
eroatoms, and their Fused Carbocyclic
Derivatives
S.M. Weinreb, R.K. Orr, in Comprehensive Heterocyclic Chemistry III, 2008

8.07.3.4 UV/Fluorescence
The fluorescence spectrum of the nonsteroidal anti-inflammatory agent piroxicam
21 has been determined in a variety of solvents (Scheme 7) <1999PCP4213>. The
key observations are that the molecule exists with a strong H-bond between the
phenolic OH and the adjacent amide. A very high Stokes shift in the excited state
was observed and attributed to the proton-transfer event (tautomerization) between
the phenolic and amide oxygens (cf. 21 → 63). In the case of protic solvents, such as
water, the open conformation 64 was observed.

Scheme 7.

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Five- and Six-membered Fused Systems

with Bridgehead (Ring Junction) Het-
eroatoms concluded: 6-6 Bicyclic with
One or Two N or Other Heteroatoms;
Polycyclic; Spirocyclic
K.C. Majumdar, S.K. Chattopadhyay, in Comprehensive Heterocyclic Chemistry III,
2008

12.14.3.5 Fluorescence and Phosphorescence Studies

The fluorescence and absorption spectra of DTT-S,S-dioxide 20a with polar covalent
bonds was studied in THF, toluene, and decalin. The spectral line and peak energy are
almost independent of the solvent polarity. The fluorescence spectra of the decalin
and toluene solutions (almost the same polarity) are red-shifted by about 5 nm, with
respect to the THF solution of higher polarity. No evident solvatochromism was
observed. The absorbance and fluorescence excitation spectra (at the fluorescence
peak wavelength) for DTT-S,S-dioxide 20a (normalized to peak value) was compared.
The fluorescence excitation signal is, in fact, dependent both on the density of the
excited state (as the absorbance) and on the efficiency of the relaxation from the
excited state of the emitting one <2005PCB6004>.

The absorption spectrum of the free DTTPP (3,5-di-

methyl-2,6-diphenyl-dithieno[3,2-b;2 ,3 -d]thiophene-4,4-dioxide 83 was com-
pared with that of DTTPP bound to antibody anti-CD3. The spectra showed that
the absorption of the fluorophore remained unaltered after conjugation, except for
some broadening of the low-energy absorption band, probably due to the increase
in the number of rotational conformers. Irradiation at exc = 404 nm led to an
intense photoluminescence signal, which retains sizeable intensity when the sample
is irradiated at exc = 480 nm. The significance of this result arises from the fact that
480 nm is the wavelength of the argon-ion laser excitation source of the currently
available commercial flow cyclometers. It was found that the activity of the antibody
was completely retained in the conjugate, both in the case of the conjugate with
antibody anti-CD8 and antibody anti-CD3 <2002CEJ5072>.

Steady-state fluorescence spectra, fluorescence quantum yield ( F) and lifetimes ( F)

of DTT 15 and DTP 23a were estimated as shown in Table 8. F for DTT is higher than
DTP. F for DTP is very small and it was difficult to estimate an accurate fluorescence
lifetime by the photon counting method due to weak fluorescence. It is noted that
the F for DTP depends largely on the solvent and is 7.7 × 10−5 in acetonitrile. This
low F value has been attributed to an addition reaction with the solvent.

Table 8. Peak positions ( F) and quantum yields ( F) of fluorescence of bithiophene

derivatives
 F (nm) F (in AN)
DTT 333 1.0 × 10−3
DTP 337 7.7 × 10−5

Phosphorescence spectra of DTT and DTP were measured in MeOH/EtOH glass at

77 K. DTT and DTP exhibited phosphorescence with clear vibrational structure. This
finding is attributed to the rigid structure caused by the bridging group at the 3,3
positions of 2,2-bisthiophene 84. The triplet energy of DTT and DTP estimated from
the O–O bands of the phosphorescence spectra are given in Table 9.

Table 9. Properties of T–T absorption bands and reaction rates of bithiophene

derivatives in triplet excited state

Triplet Enrgy/ev T (nm) T(dm3 mol−1 cm−1)

DTT 2.57 384 2.2 × 104

DTP 2.58 400 1.4 × 104

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Pyrene fluorescence to study polymeric

systems
J DUHAMEL, in Molecular Interfacial Phenomena of Polymers and Biopolymers,
2005

7.2.5 The I1/I3 ratio

The fluorescence spectrum of pyrene exhibits several emission bands between 370
and 430 nm. In the case of molecular pyrene, numerous reports have found that the
ratio of the first (I1) to the third (I3) peak responds to the polarity of the solvent.20,21
Because pyrene is a molecule, it can probe the microdomains of a heterogeneous
system at the molecular level. Consequently the I1/I3 ratio reports information
about the polarity of the specific microdomains probed by pyrene. This polarity is
sometimes referred to as micropolarity, since it is characteristic of a microdomain.
The dependence of the I1/I3 ratio with micropolarity is not as pronounced for pyrene
derivatives as that observed with the pyrene molecule. Nevertheless, the pyrenyl
pendant responds to the micropolarity of the environment when the pyrene moiety
is connected to the polymer via a single methylene unit located at the 1-position.
Longer linkers result in a loss of the response of pyrene to micropolarity.22
Measurements of the I1/I3 ratio require that the slit widths of the emission mono-
chromator of the steady-state fluorometer be kept narrow. Broad slits lead to a loss of
the resolution necessary to obtain the sharp I1 and I3 peaks required for an accurate
I1/I3 ratio measurement. It is also often found that the I1/I3 ratio does not respond
to micropolarity when the reacting group used to attach pyrene onto the polymer is
located directly onto the aromatic rings of pyrene. The methylene unit between the
reacting group and the pyrene moiety seems to be a necessary insulator to protect
the pyrene moiety from the perturbing electronic environments of many reactive
groups (carboxy, sulfoxy, amino …).

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Cis-Trans Isomerization of C=C Double

Bonds
J. Saltiel, Y.-P. Sun, in Photochromism, 2003

4.1.2 Excited singlets of isolated DPH

The fluorescence spectrum, one photon fluorescence excitation spectrum, and sin-
gle vibrational level decay kinetics have been measured for DPH seeded in helium
in a supersonic free jet (ref. 250). Consistent with observations from two photon
excitation spectroscopy in EPA glass at 77 K (ref. 217), absorption to the 21Ag state
is observed as a sharp progression of bands starting at 25,741.8 cm−1 above the
ground state (ref. 250). Fluorescence excitation intensity from the lowest observed
band, assigned as a vibronically promoted false origin, is smaller than that of the
11Bu ← 11Ag fluorescence excitation origin at 29,157 cm−1 by at least a factor of 104
(ref. 250). The relatively small shifts to 25,050 cm−1 in EPA at 77 K (ref. 217) and
to 25,187 cm−1 in n-hexane at 4.2 K (ref. 214) for the weak 21Ag ← 11Ag transition
can be contrasted with the 3200 cm−1 shift to 25,973 cm−1 in n-hexane at 4.2 K (ref.
214) experienced by the strong 11Bu − 11Ag transition, and are consistent with the
differential effects of medium polarizability change discussed above.

Especially informative is the broad shape of the fluorescence spectrum obtained

upon excitation of the 0-0 band of the 11Bu ← 11Ag transition (ref. 250). The spectrum
bears a strong resemblance to solution spectra and shows that → relaxation in the
isolated molecule is fast in the ns time scale. The fluorescence lifetime gradually
decreases with excitation frequency from 90.7 ns at 25,742 cm−1 to 39.1 ns at 30,771
cm−1. Excitation in the 0-0 band of the − 1Ag transition gives − 48.1 ns (ref.
250). Lifetimes were measured by treating decay curves 7 ns following maximum
fluorescence intensity attainment, so no kinetics information was obtained for
shorter times when rapid IVR changes must be occurring. However, the absence of
prompt fluorescence shows that relaxation to the state is rapid in the absence of
solvent effects, despite the much larger ΔEba = 9.7 kcal/mol value. It is unknown
at this time to what degree, if any, conformational changes leading to trans →
cis photoisomerization contribute as radiationless decay channels. In the absence
of fluorescence quantum yield information the lifetimes obtained provide upper
bounds for effective radiative rate constants, and clearly establish the forbidden
nature of the radiative transition.

A study of fluorescence and fluorescence excitation spectra from static vapors of DPH
(368 K) and DPO (377 K) bridges the gap between the solution work and the above
study of the jet-cooled isolated molecule. Though DPH and DPO were reported to
behave similarly, results for the former were presented in greater detail (ref. 251).
Emission from both was assigned to the 21Ag state. For DPH, fluorescence intensity
was shown to increase with increasing buffer gas pressure (perfluorohexane 0 − 250
Torr) and to decrease slightly with increasing excitation energy (310 – 340 nm). The
pressure dependence of f was fitted by assuming kfa = 4.2 × 107 s−1, independent
of excitation wavelength and buffer gas pressure. This value seems rather high in
view of the large ΔEba in the vapor phase. It corresponds to a radiative lifetime of 24
ns which is significantly smaller than actual lifetimes observed for jet-cooled DPH
(ref. 250). The identity of the radiationless channel(s) responsible for the relatively
low fluorescence quantum yields at low buffer gas pressure remains to be defined.

> Read full chapter

Cyanine Dyes
Ghodsi Mohammadi Ziarani, ... Hendrik G. Kruger, in Metal-Free Synthetic Organic
Dyes, 2018

8.2 Application of Cyanine Dyes

The fluorescence spectrums of TO derivatives 13 were studied preliminarily, and

the phenomenon of the enhancement of fluorescence was depicted. Breast cancer
cells labeled by TO derivative bearing COOH or NH2 and their derivatives were also
studied preliminarily, which offered a new try in the aspect of labeling cells by the
embedded dyes. The multiple relationships between TO derivatives and cells were
investigated. It was observed that TO–NH2 can penetrate through the cell membrane
and exist stably in the nuclear with no toxicity. Over repeated experiment, the
labeled samples showed very little photobleaching, far less than with conventional
dye molecules. So the structures of TO and its derivatives are not degraded, and the
dyes could penetrate through the cell membrane into the nucleus with no toxicity
and show fluorescence and sense information.

Dyes 18a–c having comparatively suitable solubility were chosen to determine their
transmittance properties after they were spin-coated onto glass and baked. The
difference in transmittance (%) at 450 nm of blue backlight for spin-coated dyes
18a–c before and after baking was in the range 1.1%–2.3%. Therefore, the results
clearly confirmed that the prepared dyes 18a–c were only slightly influenced by
baking during the process of fabricating the color filter, which is in accordance
with the results of the thermogravimetric analysis. Therefore, synthesized colorants
can be very suitable materials in the fabrication of LCD color filters, particularly for
blue pixels because of their extremely high thermal stability. In another study, Hua’s
group synthesized the analogues of dye 18a–c for dye-sensitized solar cells (DSSCs)
[98–102].

Selected cyanine dyes 22–28 might be suggested to be used as photosensitizers in

most polar and nonpolar organic solvents in the (blue–red) light.

The dye 33 showed an obvious loss of pink color in the presence of Hg2+ and CN−
ions, which could make it a suitable “naked eye” indicator.
Although dye 39 did not give high values in DSSC cells with low-temperature
porous ZnO, the enhanced light harvesting in the NIR region suggested that it
may be possible to increase the overall efficiency of DSSCs using visible-region
absorbing dyes through the coadsorption of a mixture of various dyes and raise the
possibility of photovoltaic windows, which could be transparent to visible light.

The possibility of growing large single crystals (>0.5 mm3) associated with a good
transparency ( max = 420 nm) of dye 48 suggested that the dye may be an interesting
material for second-order optical nonlinearities.

The dye 91 was applied to wool fabrics to give fluorescent orange shades. Color
fastness properties of dyed wool fabrics showed that the rub fastness and wash
fastness of dye 91 were both excellent (four or more), which was attributed to the fact
that dye 91 was fixed on fiber by covalent bonds. Compared with wool fabric dyed
with DEASPI, the sample using dye 91 had better color fastness to light, with a value
of 3–4. This fact may be due to the molecular structure change of dye 91 during the
dyeing process.

It was found that appropriate number of the extra electron donor and acceptor
groups in the dye structure 108 will further improve the performance of the DSSCs
based on these dyes. The longer electron lifetime can also be beneficial to improve
the power conversion efficiency of the cell, which can be achieved through the
reduction of dye aggregation during the dye sensitization.

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DI-π-METHANE HYPERSURFACES
AND REACTIVITY; MULTIPLICI-
TY AND REGIOSELECTIVITY; RE-
LATIONSHIP BETWEEN THE DI--
π-METHANE AND BICYCLE RE-
ARRANGEMENTS
HOWARD E. ZIMMERMAN, RONDA E. FACTOR, in R.B. Woodward Remembered,
1982

Emission studies magic multipliers

15The fluorescence spectrum of each compound was measured in a 4:1 methylcyclo-
hexane–isopentane soln at 77°K and 295°K using an Aminco-Keirs spectrofluorome-
ter equipped with a Hanovia 901C-1 150-W Xenon arc lamp. Concentrations of solns
were adjusted to give optical densities of 0.8–0.9. All solns were thoroughly degassed
immediately before the spectra were obtained. Emission wavelength maxima were
found to be independent of excitation wavelength over a 50 nm range. Magic
Multiplers were calculated by dividing the integrated intensity of fluorescence at
77°K by the integrated intensity of fluorescence at 295°K. The average value obtained
for each compound was: 3,3-dicarbomethoxy-1,1,5,5-tetraphenyl-1,4-pentadiene,
M = 21.0 (3 runs); 1,1-dicarbo-methoxy-3,3,5,5-tetraphenyl-1,4-pentadiene, M =
197 (3 runs).

> Read full chapter

Laser spectroscopy for medical applica-

tions
S. Svanberg, in Laser Spectroscopy for Sensing, 2014

Raman spectroscopy
In contrast to fluorescence spectra, those induced by the Raman process feature
comparatively sharp peaks due to molecular vibrations, which are normally observed
on the Stokes side of the carrier (Stokes–Raman components). A complication is that
the normally much stronger LIF process induces a broadband background on which
the Raman peaks occur. We will discuss how to abate this problem below in Sec-
 tion 14.8.1. A basic observation is that it is necessary to strongly suppress the much
stronger peak due to the elastically scattered irradiating light (the Rayleigh/Mie
component). This can be achieved with double monochromators and/or the use
of notch filters, strongly suppressing the carrier but letting close-lying radiation
through efficiently.

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