Geoscience Reference
In-Depth Information
The molecular structure can have a profound effect on the wavelengths of fluorescence
emission. The absorption properties of many biological pigments reflect the extent of con-
jugation; more specifically, when an electron in a conjugated system absorbs a photon of
light at a suitable wavelength then it can be promoted to a higher energy level. Transitions
like these are typically a
π
electron to a
π
* electron. In addition to this nonbonding elec-
trons can also be promoted (n to
π
*). Such substances are said to be extensively delocal-
ized. Conjugated systems of fewer than eight conjugated double bonds absorb exclusively
in the ultraviolet region. Each additional double bond results in the conjugated system
absorbing light at longer wavelengths (lower energy).
Equation (1.2)
states that the smaller
the energy gap between the ground and lowest electronic excited state, then the longer the
wavelengths of fluorescence. This is practically demonstrated by the fluorescence emis-
sion maxima for benzene, naphthalene, and anthracene, which are 262 nm, 320 nm, and
379 nm, respectively. With respect to biological pigments, beta-carotene has a long con-
jugated hydrocarbon chain and therefore a complex conjugated electron system, resulting
in a strong orange color. Pigments utilizing conjugation in this way range from yellow to
red. Blue and green (photosynthetic) pigments are less reliant on this type of conjugated
electron system.
Molecules or compounds that undergo electronic transitions resulting in fluorescence
are known as fluorescent probes, fluorochromes, or dyes. When such substances are con-
jugated to larger molecular structures such as nucleic acids, lignins, organic acids, and
proteins they are termed fluorophores. Fluorophores can be further divided into two broad
classes, “intrinsic” and “extrinsic” fluorophores. Intrinsic fluorophores, occur naturally
and include aromatic amino acids, porphyrins, organic acids, and green fluorescent pro-
tein. Extrinsic fluorophores include synthetic dyes (Rhodamines) or modified biochemicals
(fluorescent labeling) to produce fluorescence with known or specific properties.
1.3.4.6 The Effect of pH
The effect of pH on the fluorescence of many molecules is well known and understood. The
influence of pH is derived from the dissociation or protonation of acidic and basic func-
tional groups associated with the aromatic constituents of the fluorophores. Protonation
or dissociation can alter the chemical nature of the fluorophore in such a way that the
rates of nonradiative processes competing with fluorescence are increased or decreased.
Protonation and dissociation can result in a shift in the fluorescence emission caused by
alteration of the relative separation of the ground electronic states of the reacting mol-
ecules. For example, the protonation of electron withdrawing groups (carboxyl) results in
shifts to longer wavelengths of fluorescence, while the protonation of electron-donating
groups (amino groups) produce shifts to shorter wavelengths.
1.3.4.7 Effects of Solvents on Fluorescence Emission
The relative fluorescence intensity and spectral peak position of a molecule will vary in dif-
ferent solvents. The interactions between solvent and the solute molecules are largely elec-
trostatic, and it is usually the differences between the electrostatic stabilization energies of
