Fluorescence
Spectroscopy
Dr AKM Shafiqul Islam
School of Bioprocess
Engineering
Spectroscopy
The basis for the microscopic description of
spectroscopy is a system composed of two energy
levels. In any spectroscopy, it is necessary to induce
a transition between two energy levels to detect the
absorption/emission of electromagnetic energy. Light
is emitted or absorbed if this system is exposed to a
frequency:
E 2  E1
v
h
When we have a population of atoms or molecules
(as we always do when we do macroscopic
measurements), each state is populated with a
certain number of electrons n1 and n2 according to
Boltzmann’ distribution:
n1
 e ( E1  E2 ) / kT
n2
The energy absorbed is proportional to the
difference:
n2  n1
E  kT
 then essentially all the molecules will be in their
ground state; the radiation field will slightly perturb
this situation increasing the average number n2 and
decreasing n1 by the same amount.
E  kT
n1  n2
 the net absorption of energy will be very small
because the rate of upward transitions is equal to
the rate of downward transitions.
How Does Matter Absorb Radiation
• We see objects as colored because the transmit or
reflect only a portion of light in this region.
• When polychrometic (white light), which contains the
whole specrum of light, we see objects as colored
because certain wavelengths are absorbed while
others are reflected.
• The reflected colors impinge on our eyes, and we
perceive these colors.
Fluorescence
Thus far we have only considered absorption
processes. If resonant absorption occurs, a system
will eventually return to the ground state. There
are a number of pathways that can be followed to
return to the ground state. Important mechanisms
are radiationless transfer (molecular collisions),
fluorescent emission, and phosphorescence.
Fluorescence is probably the most important form of
spectroscopy in biology, think about GFP and all sort
of microscopies that are done now using
fluorescence as a probe of biological structure or
localization. The reason it is so widely used is that
it is a very sensitive technique (even single molecules
can be detected by fluorescence).
What is Atomic Fluorescence?
• Atomic fluorescence spectroscopy (AFS) is the
optical emission from gas-phase atoms that have been
excited to higher energy levels by absorption of
radiation.
• AFS is useful to study the electronic structure of
atoms and to make quantitative measurements of
sample concentrations.
Fluorescence Spectroscopy
In fluorescence spectroscopy, the signal being measured
is the electromagnetic radiation that is emitted from
the analyte as it relaxes from an excited electronic
energy level to its corresponding ground state. The
analyte is originally activated to the higher energy level
by the absorption of radiation in the UV or VIS range.
• Generally one to three orders of magnitude more sensitive than
corresponding absorption spectroscopy.
The Electromagnetic wave
• Electromagnetic radiation can be considered a
form of radiant that is propagated as a transverse
wave. It vibrates perpendicular to the direction of
propagation, and this imparts a wave motion to
the radiation.
• The wave is described either in terms of its
wavelength, the distance of one complete cycle,
or in terms of the frequency, passing a fixed
point per unit time. The reciprocal of the
wavelength is called wavenumber and is the
number of waves in a unit length or distance per
cycle.
Wave propagation
<380 nm
380 nm – 780 nm
Near IR: 800 nm-2,500 nm
Far IR: 15,000-100,000 nm
>780 nm
Mid-IR: 2,500-15,000 nm
Electromagnetic Wave
• The relationship between the wavelength and
frequency is
c
λ
v
• Where l is the wavelength in centimeters (cm), n
is the frequency in reciprocal seconds (s-1), or
hertz (Hz), and c is the frequency of light (3 x
1010 cm/s). The wavenumber is represented by n,
in cm-1:
1 n
n 
l c
Electromagnetic Radiation
• Electromagnetic radiation possesses a
certain amount of energy. The energy of
unit of radiation, called the photon, is
related frequency or wavelength by
E  hv 
hc
l
Where E is the energy of the photon in
ergs and h is Plank’s constant, 6.62 x
10-34 joule-second (J-s).
PHOTOLUMINESCENCE
1. Fluorescence :
Does not involve change in electron
spin; short lived (less than
microsecond). Can be observed at
room temperature in solution.
2. Phosphorescence:
Involves change in electron spin.
Long lived (seconds). Can be
observed at low temperature in
frozen or solid matrices.
3. Chemiluminescence:
Light emission due to a chemical
reaction.
SINGLET AND TRIPLET STATES
Electronic transitions responsible for absorption
spectra will give rise to singlet and triplet states and
hence to fluorescence and phosphorescence.
Singlet State: Electron spins in the ground and
excited electronic states are paired. Net spin S is
zero. Spin multiplicity 2S + 1 = 1.
Triplet State: Electron Spins in the ground and
excited electronic states are not paired. Net spin S =
1. spin multiplicity 2S + 1 = 3.
Excited states Producing Fluorescence
and Phosphorescence
• Ground Singlet State
– A molecule at room temperature normally resides
at in the ground state. The ground state is usually
singlet state (So), with all electrons paired.
• Excited Singlet State
• Excited Triplet State
Jablonski Diagram
Atomic Fluorescence
Spectroscopy
• optical emission from gas-phase atoms that
have been excited to higher energy levels
– Enhancement of sensitivity over AA
– Examine electronic structure of atoms
• Light source
– Hollow Cathode Lamp
– Laser
• Detection
– Similar to AA
Hallow cathode Tube
Fluorescence Spectroscopy
• Excitation beam
• Emission beam
Fluorescence Spectroscopy
Fluorescence Spectroscopy
PF =  (P0-P)
(1)
Where PF= radiant power of beam emitted from fluorescent cell
 = constant of proportionality (or quantum efficiency)
Beer’s law: P=P010-bc
PF=  P0 (1-10-bc)
PF=kP0c
Where k=constant of proportionality
• Linear concentration range
Factors: T, solvent, pH, impurity
(2)
(3)
(4)
Example: Pyrene
K. Kalyanasundaram and J. K. Thomas, J. Am. Chem. Soc., 1977, 99, 2039
Example: Dansyl