Geoscience Reference
In-Depth Information
Finally,
c
and
l
represent the concentration of the relevant fluorescent species and the sam-
pling path length respectively.
Both the path length and absorptivity of the species/sample is important:
α
()
λ
cl
<<
1
;(
I
λ
)
≅
K
η
(
λ
)( )(
I
λ
α
λ
)
cl
(1.17)
ex
em
em
0
ex
ex
In excitation and emission matrices the ratio of the emission intensity (
λ
em
) to the excitation
intensity (
λ
ex
) is plotted. The relationship between
λ
em
and
λ
ex
can be expressed as
I
I
( )
()
λ
λ
em
=
Kcl
ηλ αλ λ
(
)
(
−∆
)
(1.18)
em
em
ex
From this it is clear that the intensity distribution will have pronounced peaks when the
absorption maxima and the fluorescence quantum yield overlap. When a sample has a well
defined absorption maximum and a well defined quantum yield maximum, then the most
spectral structures and features are most likely to be observed when the Δ
λ
is equal to the
difference in wavelength of the emission and excitation maxima (Stoke's shift).
1.3.4.10 Scattering of Radiation
The most probable interaction between a molecule and optical radiation is Rayleigh and Mie
scattering. Both Rayleigh and Mie represent the major types of elastic scattering (negligible
energy transfer) and unlike fluorescence emission there is no wavelength shift in this pro-
cess. Rayleigh scatter involves the scattering of light by a spherical volume or entity with an
uneven refractive index. This volume or entity could manifest itself as a colloid, particle, bub-
ble, droplet, or even a density fluctuation. The size of the scattering species must, according
to Lord Rayleigh's model be much smaller (1/
λ
4
relation) than the wavelength of the incident
light. For entities and volumes equal to or about the same size as the wavelength of incident
light then Mie scattering (named after the German physicist Gustav Mie) occurs. From a
practical aspect both scattering process occur in virtually all fluorescence applications, espe-
cially in highly heterogeneous samples such as natural and waste waters. Although Rayleigh-
Mie scattering is an important natural phenomenon the scattered radiation does not bear any
identifiable signature
of the scattering species. Therefore in the application of fluorescence
spectroscopy the reduction of the Rayleigh-Mie scattered is an important consideration.
Another interaction that takes place when light interacts with molecules is Raman scat-
tering. In this process scattering takes place at discrete wavelengths governed by the size
and
symmetry of the molecule. For a nonlinear molecule of N atoms, there will be differ-
ent possible scattering wavelengths or modes (3N - 6). Some of these modes may give
overtones and combination modes, thereby creating a series of modes for each molecular
species. The fundamental modes correspond to the energy levels of the vibrational levels
and the wavelength of a particular Raman mode,
λ
r
, is given by
11
λλ
ν
i
−=∆
(1.19)
r
