Biomedical Engineering Reference
In-Depth Information
2.2.3.2 Substituent Effects: (Example—Electron-Donating and Electron-Withdrawing
Groups)
Electron-donating substituents on the parent ring system lead to an increase in molar
absorptivity and a shift in both the fluorescence excitation and emission spectral profiles
(17). Electron-withdrawing substituent effects are difficult to predict and depend on
whether the carbonyl group is an aldehyde, ketone, or carboxylic acid. It also depends on
the nature of aromatic carbonyl compound and solvent polarity. Nitroaromatics are usu-
ally phosphorescent, and if they are fluorescent, the fluorescence is usually not observed
due to internal conversion caused by the charge-transfer efficiency of the excited state (17).
2.2.3.3 Heterocyclic Compounds
Fluorescence of aromatics that contain one or more heterocyclic nitrogens (azarenes)
depends heavily on solvent polarity (17). Related heterocycles containing oxygen and sul-
fur can be similarly compared, and in the case where heterocycles contain N, O, or S atoms
with single bonds to carbon, the quantum yield is relatively high owing to the orientation
of the ? electron system of the rings and the nonbonding orbital of the N, O, or S atom (17).
Several fluorophores of practical use are heterocyclic and contain either an N, O, or S atom
or some combination of the three. Coumarins, rhodamines, pyronines, fluoresceins, cya-
nine, and oxazine classes are examples of some of the more common classes of heterocyclic
fluorophores (17).
2.2.3.4 Temperature
An increase in temperature will decrease the quantum yield of fluorescence, as the nonra-
diative thermal processes owing to the increase in random molecular motion and
increased average kinetic energy such as collision and intramolecular vibrations and rota-
tions are enhanced as temperature increases. If the nonradiative processes are now higher,
this will contribute to a lower quantum yield of detectable fluorescence.
2.2.3.5 Charge Transfer and Internal Rotation
Photoinduced intramolecular charge transfer occurs as a result of the excitation of an electron
from a lower lying molecular orbital to a higher energy state molecular orbital (usually
HOMO to LUMO) transition (17). If the excited-state molecular orbital is sufficiently
separated in space from the ground-state molecular orbital, this causes an instantaneous
change in the dipole moment of the fluorescent molecule (17). This change is not in equilib-
rium with the surrounding solvent molecules and has an enhanced effect as polarity of the
solvent increases (17). In a fluid medium, the solvent molecules rotate within the excited-state
lifetime of the fluorophore such that an organized solvent shell that is in equilibrium with the
fluorophore is developed (17). This leads to a relaxed intramolecular charge transfer state (17).
The relaxation caused by the solvent organization leads to an increased red shift of the fluo-
rescence emission of fluorophores as the solvent polarity is increased (17). The intramolecu-
lar charge transfer may also be accompanied by internal rotation within the fluorophore
under certain conditions (17). In some cases, the internal rotation and intramolecular charge
transfer processes accompanied by an increase in solvent polarity will stabilize the excited
state and lead to fluorescence emission. However, in other cases the internal rotation can
occur without solvent relaxation and can lead to a twisted intramolecular charge transfer
species that is not fluorescent (17). Photoisomerization can also occur where rotation about
double bonds takes place and leads to nonradiative deexcitation in nonpolar solvents.
However, photoisomerization in polar media is inefficient owing to the stabilization of the
intramolecular charge transfer species, which leads to a radiative fluorescence emission (17).
