Post on 01-Jan-2017
Chapter 6
Molecular Fluorescence
and Phosphorescence
Luminescence can be classifieds according
to the source of excitation into:
1. Photoluminescence: deactivation takes
place after excitation with photons
2. Radioluminescence: ground state
molecules are excited by collisions with
high energy particles
3. Chemluminescence: ground state
molecules are excitted by certain
chemical reactions
Sources of Luminescence
Characteristics of Photoluminescence
Fluorescence is short-lived with luminescence ending almost
immediately.
Phosphorescence involves change in electron spin and may
endure for several seconds.
In most cases, photoluminescent radiation tends to be at
longer wavelengths than excitation radiation.
Chemiluminescence is based on an excited species formed
by a chemical reaction.
Types of Fluorescence/phosphorescence
• Resonance radiation (or fluorescence) – absorbed radiation
is reemitted without alteration.
• More often, molecular fluorescence (phosphorescence) occurs
as bands centered at wavelengths longer than resonance
line. This shift to longer wavelengths is Stokes shift.
Excitation and de-excitation process
Molecular Multiplicity, M
M = 2S + 1
S = spin quantum number of the molecule
= net spin of the electrons in the molecule
•Most organic molecules have S = 0 because molecules
have even number of electrons thus the ground state
must have all electrons paired
•M = 2 X (0) + 1 = 1; Molecules in the ground state
mostly have a singlet state, So. S1 and S2 for first and
second excited states
• While molecules in the excited state, one e- may
reverse its spin
• S = (+1/2) + (+1/2) = 1
M = 2(1) + 1 = 3 = Triplet State= T1
• A molecule with an even number of e- can not
have a ground triplet state because the spins of
all electrons are paired
• Molecules with one unpaired electron are in
doublet state (organic free radicals)
Spin Orientations
• The allowed absorption process will
result in a singlet state.
• A change in electron spin is, technically,
a "forbidden" process
•“Forbidden" process according to
quantum mechanics means unlikely, not
“ absolutely can’t happen”
Electronic States
Singlet State: electron spins paired, no splitting of energy level.
May be ground or excited state.
Doublet State: free radical (due to odd electron).
Triplet State: one electron excited to higher energy state, spin
becomes unpaired (parallel).
Difference between triplet and singlet states
1. Molecule is paramagnetic in the T excited state and
diamagnetic in the S excited state
2. S T transitions (or reverse) are less probable
than S S transitions
Thus average lifetime of T excited state (10-4 s) is
longer than the S excited state (10-5 - 10-8 s)
Also absorption peaks due to S-T transitions are
less sensitive than S-S transitions
When an excited triplet state can be populated from
an excited S state of certain molecules, a
phosphorescence process will be the result
Energy of a Molecule (Jablonski energy-level diagram)
Energy Levels for Luminescence Transitions
+quenching
S0
S1
T0
transition involving
emission/absorption of
photon
radiationless transition
ab
so
rpti
on
+hν
flu
ore
scen
ce
-hν
inte
rnal
co
nvers
ion
inte
rsyste
m
cro
ssin
g
inte
rnal
co
nvers
ion
Fluorescence in the Jablonski energy-level diagram
Interpretation of the Energy Diagram
• Absorption : Ground state to Excited state
• (10-15 sec)
• Relaxation: Excited state to Ground state
– Internal Conversion (IC)
• nonradiative (thermal, collisional) relaxation of
electrons through vibrational states (10-12 - 10-14 sec)
– Emission
• fluorescence (spontaneous emission: 10-10 - 10-8 sec)
• phosophorescence (10-3 - 10-0 sec)
– phosphorescence requires intersystem crossing
(flip of electron spin)
» Ground state singlet
» Excited state singlet
» Spin flip (now in Triplet state)
» intersystem crossing
» Need another Spin flip to be allowed to go
back to Ground state singlet
– Once in the triplet state, de-excitation to the
ground singlet state is “forbidden”.
• Consequently, the molecule "hangs" in
the triplet state for a considerably longer
period of time than it would otherwise.
When the emission finally comes, it is
called phosphorescence.
Deactivation Processes
-Internal Conversion IC
-Inter System Crossing ISC
- Quenching
- Fluorescence
- Phosphorescence
The molecule can rapidly dissipate excess
vibrational energy as:
1. heat by collision with solvent molecules through
vibrational relaxation process
2. EMR
Rates of Absorption and Emission
• The rate at which a photon of radiation is
absorbed is enormous, the process
requiring on the order o f 10-14 to 10-15s.
• Fluorescence emission, on the other hand,
occurs at a significantly slower rate.
– Here, the lifetime of the excited state is
inversely related to the molar absorptivity of
the absorption peak corresponding to the
excitation process.
• The favored route to the ground state is the one that minimizes the lifetime of the excited state.
• Thus, if deactivation by fluorescence is rapid with respect to the radiationless processes, such emission is observed.
• On the other hand, if a radiationless path has more favorable rate constant, fluorescence is either absent or less intense.
Vibrational Relaxation
• This relaxation process is so efficient that
the average lifetime of a vibrationally excited
molecule is 10-12s or less, a period
significantly shorter than the average lifetime
of an electronically excited state.
Internal Conversion
• The term internal conversion describes
intermolecular processes by which a
molecule passes to a lower energy electronic
state without emission of radiation.
• These processes are neither well defined nor
well understood, but it is apparent that they
are often highly efficient, because relatively
few compounds exhibit fluorescence
Predissociation
• As a result if internal conversion, electron
may move from a higher electronic state to
an upper vibrational level of a lower electronic
state in which the vibrational energy is enough
to cause rupture of a bond
• In a large molecule there is an appreciable
probability for the existance of bonds with
sterngths less than the electronic excitation
energy of the chromophores
Dissociation
• The absorbed radiation excites the electron of
a chromophore directly to a sufficiently high
vibrational level to cause rupture of the
chromphoric bond. That is no internal
conversion is involved.
• Dissociation processes also competes with the
fluorescent process
External Conversion
• Deactivation of an excited electronic state may involve interaction and energy transfer between the excited molecule and the solvent or other solutes.
• These processes are called collectively external conversion, or collisional quenching.
• Evidence for external conversion includes the marked effect upon fluorescence intensity exerted by the solvent; furthermore, those conditions that tend to reduce the number of collisions between particles generally lead to enhanced fluorescence.
• Intersystem crossing takes place from excited
singlet to excited triplet state.
• Transition occurs between the singlet ground
state (electrons are anti-parallel & paired) to an
excited state(electrons are parallel
andunpaired)
• Return to ground state is much slower process
than fluorescence, or Phosphorescence.
• Emitted radiation is of an even longer
wavelength because the energy difference
between the two is small.
Intersystem crossing
Fluorescence
De-excitation can occur via a
radiative decay, i.e. by spontaneous
emission of a photon. The radiative
de-excitation process can be
described as a monomolecular
process:
The vibrational relaxation of any
electronic state is always much faster
than photon emission. Therefore, all
observed fluorescence normally
originates from the lowest vibrational
level of the electronic excited state.
Electronic
ground state
Electronic
excited state
en
erg
y
v=0
excFexc nk
dt
dn
v=0
Fluorescence
Furthermore, the shape of the
emission spectrum is approximately
the mirror image of the absorption
spectrum, providing that the ground
and excited state have similar
vibrational properties.
Electronic
ground state
Electronic
excited state
en
erg
y
v=0
v=0
Most of the fluorescence spectrum is shifted to lower energies
(longer wavelengths), compared to the absorption spectrum.
Mirror Image Spectra
The above spectra are plotted as amplitude versus wave number. When plotted versus wavelength the mirror effect
is not as pronounced.
• The shortest
wavelength in the
fluorescence
spectrum is the
longest wavelength
in the absorption
spectrum
Phosphorescence
• Deactivation of electronic excited states may
also involve phosphorescence.
• After intersystem crossing to a triplet state,
further deactivation can occur either by internal
or external conversion or by phosphorescence.
• External and internal conversions compete so
successfully with phosphorescence that this
kind of emission is ordinarily observed only at
low temperatures, in highly viscous media or by
molecules that are adsorbed on solid.
Phosphorescence
Phosphorescence occurs when a
“forbidden” spin exchange converts
the electronic excited singlet state
into a triplet state:
The triplet state relaxes rapidly to the
v=0 vibrational level, which has lower
energy than the corresponding
excited singlet state. The transition to
the electronic ground singlet state
with the emission of a photon is spin-
forbidden. Therefore the molecule
gets trapped in the triplet state.
Electronic
ground state
Electronic
excited state
en
erg
y CrossingmIntersyste
Phosphorescence
In practice, the emission of a photon
and the recovery of the ground state
occurs, but with low efficiency.
Since the triplet state has generally
lower energy than the excited singlet,
phosphorescence occurs at longer
wavelengths (lower frequencies) and
can easily be distinguished from
fluorescence. The de-excitation of
molecules due to phosphorescence is
described by:
Electronic
ground state
Electronic
excited state
en
erg
y
excISexc nk
dt
dn
Phosphorescence
Being spin-forbidden, the transition
from the excited triplet to the ground
singlet occurs very slowly, with a
radiative lifetime in the order of
seconds, or longer.
Phosphorescence can be observed
only when other de-activating
processes have been suppressed,
typically in rigid glasses, at low
temperature and in the absence of
oxygen.
In solution other de-excitation
processes, such as quenching are
much more efficient, and therefore
phosphorescence is rarely observed.
Quenching
•Energy gets transferred to the quencher, usually
through collisions with a nearby residue or molecule
•This reduces photon emissions and decreases
fluorescence intensity.
Quenching
•Two processes can diminish amount of light energy emitted
from the sample:
•Internal quenching due to intrinsic structural feature e.g.
structural rearrangement.
•External quenching interaction of the excited molecule
with another molecule in the sample or absorption of
exciting or emitted light by another chromophore in
sample.
•All forms of quenching result in non-radiative loss of
energy.
Quenching
De-excitation can result from collisions with other solute
molecules (Q), capable of accepting the excess energy and
therefore of quenching the excited states:
exchquenchquencherexchquenchexc nknnk
dt
dn
excQ
QgroundQexc
'
][][
*
Usually Q is in large molar excess over the excited state and the
observed kinetic is a pseudo-first order. Oxygen is an efficient
quencher, with quenching rates limited basically by diffusion. At
millimolar oxygen concentration this means
19s10~'
quenchk
Rate Constants and Quenching
• The rate constant for fluorescence is roughly proportional to the molar absorptivity
e 104 103 102
kf 109 108 107
• The rate constant for intersystem crossing depends upon the singlet-triplet gap, the smaller the gap the larger the rate constant • The rate constant for intersystem crossing is increased with Br and I substitution into the double bond structure • During the lifetime of the excited state a molecule can
loose energy via collisions, this is called quenching
1
* *0 0
*1 0
q
q
k
k
S Q S Q S Q heat
S Q T Q
common quenchers are oxygen, molecules with heavy atoms, and molecules with unpaired spins
Kinetics of Fluorescence and Phosphorescence
Intensity of absorbed light = I = Io - IT
Where I is known also as Rate of absorption
That is exactly equal rate of deactivation
I = (kIC + kISC + kf + kQ [Q]) [S1]
kIC + kISC + kf + kQ are the first-order rate constants
of the corresponding deactivation processes. kQ is
the second-order quenching rate constant,
[Q] is the quencher concentration
[S1] is the concentration if S1 molecules
Vibrational relaxation has been included in kIC
Efficiency of fluorescence is measured
in terms of the fluorescence quantum
yield, f
f = # of photons emitted
# of photons absorbed
Rate of fluorescence= If = I f = kf[S1]
= f (kIC + kISC + kf + kQ [Q]) [S1]
f = kf / (kIC + kISC + kf + kQ [Q])
Fluorescence Quantum Yield
• The higher the value of f the greater will be
the observed fluorescence.
If the rate constants relative to other de-
excitation processes are small compared to kf
then the compound will have a value of f ~ 1.
So by definition a non-fluorescent compound
has a value of f = 0, where all energy
absorbed by the molecule is lost via non-
radiative processes such as collisional
deactivation.
• The quantum yield of a compound is usually
determined relative to a standard for which f is
already known.
• The intensity of fluorescence of a fluorophore is
referred to as brightness: the higher this is, the more
extinction coefficient (e) and the quantum yield (f ).
• f allows a qualitative interpretation of many of the
structural and environmental factors that affect
fluorescent intensity
• The variables that lead to higher kf values and lower
values to the other k terms will enhance fluorescence
To obtain a large quantum yield: find a molecule with a large molar absorptivity
substitute a highly symmetric molecule with a
group having a lone pair of electrons (-OH or –
NH2)
keep oxygen and free radicals out of the solution
don't use molecules with heavy halogens
ratio naphthalene 1-fluoro 1-chloro 1-bromo 1-iodo p/f 0.093 0.068 5.2 16.4 >1000
The lifetime of the S1 state is given by:
= 1/ (kIC + kISC + kf + kQ [Q])
If all processes competing with fluorescence
are absent, then
r (radiative lifetime) = 1 / kf
Thus,
f = / r
For Phosphorescence p = 1/ (kp + k’VR + kQP [Qp]
and p / t = P / PR
Kp = First order decay const of T1 to S0 state
k’VR = const. For vibrational relaxation of the
T1 state
kQP [Qp] = pseudo first-order rate const. For
quenching of the triplet state by impurity
quincher, Qp
P and PR = lifetimes in, respectively, the
presence and absence of the competitive
radiationless processes
t = efficiency of formation of the triplet
state
Effect of Concentration on Fluorescent Intensity
If = I f = f (Io – IT) ….(1)
IT = Io X 10 -ebc …… (2)
Where e is the molar absorptivity of the
fluorescing molecule. Substituting Eq 2 in Eeq 1
If = fIo (1– 10 -ebc ) …. (3)
The exponential term in Eq 3 can be expanded as
a Maclaurin series to
If= fIo [2.303 ebc - (2.303 ebc )2/2! +(2.303 ebc )3/3!.. )
Provided 2.303 ebc < 0.05, all of the subsequent
terms in the brackets become small with respect to
the first. Thus, we may write
If= 2.303 fIo ebc
Or If = kC.
If VS C is straight line at low concentration
Factors responsible for non linearity
1. The concentration: When 2.303 ebc is more than 0.05,
the linearity is lost
2. Self quenching: collisions between excited molecules
3. Self absorption: When the wave length of emission
overlaps an absorption peak. Fluorescence is then
decreased as the emitted beam traverse the solution
Excitation and Emission Spectra
Fluorescing molecules are characterized by two types
of spectra:
1. Excitation Spectrum:
Fluorescence intensity is observed as a function of
exciting at some fixed emission
2. Emission (Fluorescence and phosphorescence) spectrum:
Emission intensity is measured as a function of emitted
at fixed exciting
3. Emission spectrum is usually used for analytical
applications
4. Excitation spectrum is run first to confirm the identity of
the substance
5. Fluorescence Spectrum occurs at longer than does the
excitation (absorption) spectrum
6. Only the longer band of absorption and the shorter
band of fluorescence will generally overlap
7. Since the vibrational spacing in the ground state So
and the first excited singlet state S1 will often be similar
for large molecules the fluorescence spectrum is often
mirror image of the absorption spectrum
8. Because phosphorescence emission occurs from the
triplet state there is no mirror relationship with the
absorption band of the lowest excited singlet
9. Since emission almost always occurs from the first
excited state, the emission spectrum is independent of
of excitation
10. Since the quantum yield of emission is generally
independent of of excitation thus the excitation
spectrum is independent of the emission monitored
• In order to scan the two types of spectra, tow monochromators
are used: Excitation monochromator and Emission
monochromator
• Excitation spectrum is recorded when the emission monochrom.
is set at fixed max (fluor. or phosph.) and the
excitation monochromator is allowed to vary.
It is used when the compound to be studied for the first
time
• Emission spectrum is recorded when the excitation
monochromator is set at a fixed (max of absorption)
and the emission monochromator is allowed to vary
(This is usually used for analytical purposes)
Fluorescent Excitation and Emission Spectra
Fluorescent Excitation and Emission Spectra
Excitation Spectrum
Observe Emission at
single wavelength while
scanning excitation wavelengths
Emission Spectrum
Observe Emission spectrum
while keeping excitation
at a single wavelength
Sample Spectra
Excitation (left), measure luminescence at fixed wavelength while varying
excitation wavelength. Fluorescence (middle) and phosphorescence (right),
excitation is fixed and record emission as function of wavelength.
Electronic Transition Types in Fluorescence
• Seldom to have fluorescence by absorbing Uv at < 250 nm
At this range of deactivation of excited state may take
place by predissociation (Rupture of bonds after IC) or
dissociation (bond rupture after absorption)
Thus, Fluorescence due to * - transition is seldom
observed
• Fluorescence is limited to the less energetic * - and
* - n transitions depending upon which is less energetic
• Fluorescence most commonly arises from transition from
the first excited state to one of the vibrational levels of the
ground state.
Quantum Efficiency and Transition Type
• f (* - ) > f (* - n) transition
e for * - transition is 100 – 1000 fold greater and
this is a measure for transition probability
Thus, the lifetime of * - is shorter than * - n
and kf is larger
• The rate constant for ISC is smaller for * -
because the energy difference for singlet/triplet states
is larger. That is more energy is required to unpair
the electrons of the * excited state. Thus, overlap of
the triplet vibrational levels with those of the singlet
state is less and the probability of ISC is smaller
•In Summary:
Fluorescence is more commonly associated with * -
transition state because:
1. * - transitions possess shorter average lifetime
2. Deactivation processes that compete with fluorescence
are less likely to occur
• Fluorescence is favored when
1. Energetic difference between the excited singlet state
and triplet state is relatively large
2. Energetic difference between the first excited state
and the ground state is sufficiently large to prevent
appreciable relaxation to the ground state by
radiationless processes
Variables that Affect Fluorescence
•Structure and structural Rigidity
•Temperature – increased temperature, decreased quantum yield
•Solvent Viscosity – lower viscosity, lower quantum yield
•Fluorescence usually pH-dependent
•Dissolved oxygen reduces emission intensity
•Concentration:
Self-quenching due to collisions of excited molecules.
Self-absorbance when fluorescence emission and absorbance
wavelengths overlap.
Fluorescence And Structure
• The most intense and the most useful fluorescence is found in compounds containing aromatic functional groups with low-energy to * transition levels.
• Compounds containing aliphatic and alicyclic carbonyl structures or highly conjugated double-bond structures may also exhibit fluorescence,
• Most unsubstituted aromatic hydrocarbons fluoresce in solution; the quantum efficiency usually increases with the number of rings and their degree of condensation.
• The simple heterocyclics, such as pyridine, furan, thiophene, and pyrrole do not exhibit fluorescence; on the other hand, fused ring structures ordinarily do.
• With nitrogen heterocyclics, the lowest-energy electronic transition is believed to involve n to * system that rapidly converts to the triplet state and prevents fluorescence.
• Fusion of benzene rings to a heterocyclic nucleus, however, results in an increase in the molar absorptivity of the absorption peak. The lifetime of an excited state is shorter in such structures; fluorescence is thus observed for compounds such as quinoline, isoquinoline, and indole.
• Substitution of a carboxylic acid or carbonyl group on an aromatic ring generally inhibits fluorescence.
• In these compounds, the energy of the n to * transition is less than that of the to * transition; as pointed out earlier, the fluorescence yield from the former type of system is ordinarily low
Heavy Atom Effect
• Halogens constituents cause a decrease in fluorescence and the decrease increases with atomic number of halogens
• The decrease in fluorescence with increasing atomic number of the halogen is thought to be due in part to the heavy atom effect, which increases the probability for intersystem crossing to the triplet state.
• Spin/orbital interactions become large in the presence of heavy atoms and a change in spin is thus more favorable
• Predissociation is thought to play an important role in iodobenzene (for example) that has easily ruptured bonds that can absorb the excitation energy following internal conversion.
• Substitution of a carboxylic acid or carbonyl group on an aromatic ring generally inhibits fluorescence. In these compounds, the energy of the n,* transition is less than that of the , * transition.
• The electromagnetic fields that are associated with relatively heavy atoms affect electron spins within a molecule more than the fields associated with lighter atoms.
• The addition of a relatively heavy atom to a molecule causes excited singlet and triplet electrons to become more energetically similar. That reduces the energetic difference between the singlet and triplet states and increases the probability of intersystem crossing and of phosphorescence. The probability of fluorescence is simultaneously reduced.
• The increased phosphorescence and decreased fluorescence with the addition of a heavy atom is the heavy-atom effect.
• If the heavy atom is a substituent on the luminescent molecule, it is the internal heavy-atom effect. The external heavy-atom effect occurs when the heavy atom is part of the solution (usually the solvent) in which the luminescent compound is dissolved rather than directly attached to the luminescent molecule.
• The effect that the halides have upon a luminescent molecule is an example of the internal heavy-atom effect.
• If a heteroatom exists in a luminescent molecule,
the transition from the ground state to the first
excited singlet state can be an n to * transition.
• Electron in a nonbonding orbital that is
associated with the heteroatom is excited to a *
orbital of the molecule.
• Molar absorptivities associated with n to *
transitions are usually relatively small (less than
1000) in comparison with absorptivities
associated with to * transitions because
nonbonding n orbitals do not overlap with *
orbitals as much as bonding orbitals do.
• Consequently, less fluorescence generally is
observed following excitation by an n to *
transition than is observed following excitation
by a to * transition
Fluorescence and Structure
Factors That Affect Photoluminescence
• Photoluminescence is favored when the absorption is efficient (high absorptivities).
• Fluorescence is favored when
1. the energetic difference between the excited singlet
and triplet states is relatively large
2. the energetic difference between the first excited singlet state and the ground state is sufficiently large to prevent appreciable relaxation to the ground state by radiationless processes.
• Phosphorescence is favored when
1. the energetic difference between the first excited singlet state and the first excited triplet state is relatively small
2. the probability of a radiationless transition from the triplet state to the ground state is low.
• Any physical or chemical factor that can affect any of the transitions can affect the photoluminescence.
• These factors include: structural rigidity, temp., solvent, pH, dissolved oxygen.
Effects of structural rigidity
• Photoluminescent compounds are those compounds in which
the energetic levels within the compounds favor de-excitation
by emission of uv-visible radiation rather than by loss of
rotational or vibrational energy
• Fluorescing and phosphorescing compounds usually have a
rigid planar structure
• the quantum efficiencies for fluorene and biphenyl are nearly
1.0 and 0.2, respectively, under similar conditions CH2 causes
more rigidity
• The rigidity of the molecule prevents loss of energy through rotational and vibrational energetic level changes.
• Any subsistent on a luminescent molecule that can cause increased vibration or rotation can quench the fluorescence.
• The planar structure of fluorescent compounds allows delocalization of the -electrons in the molecule. That in turn increases the chance that luminescence can occur because the electrons can move to the proper location to relax into a lower energy localized orbital.
• Organic compounds that contain only single bonds between the carbons do not luminesce owing to lack of absorption in the appropriate region and lack of a planar and rigid structure.
• Organic compounds that do luminesce generally consist of rings with alternative single and double bonds between the atoms (conjugated double bonds) in the rings.
• The sp2 bonds between the carbons in the rings cause the desired planar structure, and the alternating double bonds give rigidity and provide the -electrons electrons necessary for luminescence.
Temperature Effect
• The quantum efficiency of fluorescence in most
molecules decreases with increasing temperature
• Due to increased frequency of collisions at elevated
temperatures the probability for deactivation by
external conversion is improved.
Solvent Effect
• A decrease in solvent viscosity also increases the likelihood of external conversion and leads to the decrease in quantum efficiency
• The fluorescence of a molecule is decreased by solvents containing heavy atoms or other solutes with such atoms in their structure; carbon tetrabromide and ethyl iodide are examples.
• The effect is similar to what occurs when heavy atoms are substituted into fluorescing compounds; orbital spin interactions result in an increase in the rate of triplet formation and a corresponding decrease in fluorescence.
• Compounds containing heavy atoms are frequently incorporated into solvents when enhanced phosphorescence is desired.
Effect of pH on Fluorescence
• Fluorescence of an aromatic compound with acidic ring substituents is usually pH-dependent.
• Both and the emission intensity are likely to be different for the ionized and nonionized forms of the compound.
• The data for phenol and aniline shown illustrate this effect.
• The changes in emission of compounds of this type arise from the differing number of resonance species that are associated with the acidic and basic forms of the molecules.
• The additional resonance forms lead to a more stable first excited state; fluorescence in the ultraviolet region is the consequence.
• Thus, close control of pH is required for fluorescence studies
Effect Of Dissolved Oxygen
• The presence of dissolved oxygen often reduces the intensity of fluorescence in a solution.
• This effect may be the result of a photochemically induced oxidation of the fluorescing species.
• More commonly, however, the quenching takes place as a consequence of the paramagnetic properties of molecular oxygen, which promotes intersystem crossing and conversion of excited molecules to the triplet state.
• Other paramagnetic species also tend to quench
fluorescence.
Fluorescence and
Phosphorescence Instruments
Design luminescence instruments
• Filter fluorometers (fluorometers, flurimeters)
and filter phosphorimeters
Work at fixed exc and fixed emi
• Spectrofluorometers & spectrophophorimetrs
Capable of scanning. Two monochromators
are required
Features of Fluorescence and
Phosphorescence Instruments
• Almost same components as Uv-Vis instruments
• Most of them are double beam configuration to allow
compensation of power source fluctuations
• Though fluorescence is propagated in all directions
the most convenient one is that at right angles to the
excitation beam.
– At other angles scattering from solutions and cell walls
may become appreciable
• The use of attenuator helps reducing the power of
the reference beam to approximately that of the
fluorescent radiation beam
Components of Fluorometers and Spectrofluorometers
Sources • A source that is more intense than the tungsten or deuterium
lamps employed for Uv-Vis.
• The magnitude of the output signal, and thus the sensitivity, is directly proportional to the source power Po.
• A mercury or xenon arc lamp is commonly employed
• The most common source for filter fluorometers is a low-pressure mercury-vapor lamp equipped with a fused silica window.
• This source produces intense lines at 254, 366, 405, 436, 546, 577, 691, and 773 nm. Individual lines can be isolated with suitable absorption or interference filters.
• Various types of lasers were also used as excitation sources for photoluminescence measurements.
• Tunable dye laser employing a pulsed nitrogen laser as the primary source. Monochromatic radiation between 360 and 650 nm is produced.
Filters And Monochromators
• Both interference and absorption filters
have been employed in fluorometers.
• Most spectrofluorometers are equipped
with grating monochromators.
DETECTORS
(Transducers)
• Luminescence signals are of low
intensity thus, large amplifier gains
are required
• Photomultiplier tubes
• Diode-array detectors
• Cooling of detector is used
sometimes to improve S/N ration
Cells and Cell Compartments
• Both cylindrical and rectangular cells
fabricated of glass or silica are employed for
fluorescence measurements.
• Care must be taken in the design of the cell
compartment to reduce the amount of
scattered radiation reaching the detector.
Baffles are often introduced into the
compartment for this purpose.
Instrument Designs: Fluorometers
• The source beam is split near the source into a reference beam and a sample beam.
• The reference beam is attenuated by the aperture disk so that its intensity is roughly the same as the fluorescence intensity.
• Both beams pass through the primary filter, with the reference beam then being reflected to the reference photomultiplier tube.
• The sample beam is focused on the sample by a pair of lenses and causes emission of fluorescent radiation.
• The emitted radiation passes through a second filter and then is focused on the second photomultiplier tube.
• The electrical outputs from the two detectors are fed into a solid state comparator, which computes the ratio of the sample to reference intensities; this ratio serves as the analytical parameter.
Nearly all fluorometers (spectrofluorometers) are
double-beam systems.
Spectrofluorometer
Fluorometer or Spectrofluorometer
Filter Fluorometer
Spectrofluorometers
• spectrofluorometers are capable of providing both excitation and emision pectra.
• The optical design of one of these, which utilizes two grating monochromators, is shown above
• Radiation from the first monochromator is split, part passing to a reference photomultiplier and part to sample.
• The resulting fluorescence radiation, after dispersion by the second monochromator, is detected by a second photomultiplier.
• The emission spectra obtained will not necessarily compare well with spectra from other instruments, because the output depends not only upon the intensity of fluorescence but also upon the characteristics of the lamp, detector, and monochromators.
• All of these instrument characteristics vary with wavelength and differ from instrument to instrument.
• A number of methods have been developed for obtaining a corrected spectrum, which is the true fluorescence spectrum freed from instrumental effects; many of the newer and more sophisticated commercial instruments provide a means for obtaining corrected spectra directly
Observe Fluorescent Excitation
and Emission Spectra Simultaneously
Spectrofluorometer based on Array Transducers
Transducer is a
two-dimensional
device that sees
the excitation and
emission radiation
in two planes
Phosphorimeters & Spectrophosporimeters
• Instruments that have been used for studying
phosphorescence are similar in design to the
fluorometers and spectrofluorometers just
considered, except that two additional components
are required
1. Excitation must be gated in time to observe
phosphorescence in the absence of fluorescence
emission
– A device that will alternately irradiate the sample and, after a
suitable time delay, measure the intensity of
phosphorescence.
– The time delay is required to differentiate between long-lived
phosphorescence and short lived fluorescence that would
originate from the same sample
2. Ordinarily, phosphorescence measurements
are performed at liquid nitrogen temperature
(-196oc) in order to prevent degradation of the output by collisional deactivation (quenching).
• Quenching effects are usually competitive enough to prevent phosphorescence observation at room temperatur
• Thus, as shown in the Figure, a Dewar flask with quartz windows is ordinarily a part of a phosphorimeter.
• At the temperature used, the analyte exists as a solute in a glass of solid solvent (a common solvent is a mixture of diethylether, pentane, and ethanol).
Phosphorimeters
Rotating can and Dewar flask are used.
Dewar is placed inside the rotating can that has two slits.
As the slit moves into line with excitation beam the sample is excited. The
speed of rotation is such that short lived fluorescence is ceased before
the slit moves into line with the emission detector so that only
phosphoriscence is observed.
Applications of Photoluminescence Methods
• Fluorescence and phosphorescence methods are applicable to lower concentration ranges and are among the most sensitive analytical techniques
• The enhanced sensitivity arises from the fact that the concentration-related parameter for fluorometry and phosphorimetry can be measured independent of the power of the source Po.
• The sensitivity of a fluorometric method can be improved by increasing Po or by further amplifying the fluorescence signal. In spectrophotometry, in conrast, an increase in Po results in a proportionate change in P and therefore fails to affect A.
• The precision and accuracy of photoluminescence methods are usually poorer than those of spectrophotometric procedures by a factor of perhaps two to five.
• Generally, phosphorescence methods are less precise than their fluorescence counterparts.
Fluorometric Determination of Inorganic Species
• Inorganic fluorometric methods are of two types.
1. Direct methods involve the formation of a
fluorescent chelate and the measurement of its
emission.
2. A second group is based upon the diminution of
fluorescence resulting from the quenching action
of the substance being determined.
• The latter technique has been most widely used for anion analysis.
Two factors greatly limit the number of transition-metal ions that
form fluorescing chelates.
1. Many of these ions are paramagnetic; this property increases the rate of intersystem crossing to the triplet state. In solution most T states lose all of their electronic energy by collisional deactivation or by rapid conversion to their So state without emitting a photon. Thus paramagnetic metal ions (Fe3+, Co2+, Ni2+ and Cu2+) quench the fluorescence of their chelates.
2. Transition-metal complexes are characterized by many closely spaced energy levels, which enhance the likelihood of deactivation by internal conversion.
• Nontransition-metal ions are less susceptible to the foregoing deactivation processes; it is for these elements that the principal inorganic applications of fluorometry are to be found.
• It is noteworthy that nontransition-metal cations are generally colorless and tend to form chelates that are also without color. Thus, fluorometry often complements spectrophotometry.
Cations that form Fluorescing Chelates
FLUOROMETRIC REAGENTS
• The most successful fluorometric reagents for cation analyses
have aromatic structures with two or more donor functional
groups that permit chelate formation with the metal ion.
Fluorometric Determination of Organic Species
• They are used for a wide variety of organic compounds, enzymes and coenzymes, medicinal agents, plant products, steroids and vitamins.
• It is important for Food products, pharmaceuticals,
clinical samples, and natural products.
Applications of Phosphorimetric Methods
• Phosphorescence and fluorescence methods tend to be complementary, because strongly fluorescing compounds exhibit weak phosphorescence and vice versa.
• " For example, among condensed-ring aromatic hydrocarbons, those containing heavier atoms such as halogens or sulfur often phosphoresce strongly; on the other hand, the same compounds in the absence of the heavy atom tend to exhibit fluorescence rather than phosphorescence.
• Phosphorimetry has been used for determination of a variety of organic and biochemical species including such substances as nucleic acids, amino acids, pyrine and pyrimidine, enzymes, petroleum hydrocarbons, and pesticides.
• However, perhaps because of the need for low temperatures and the generally poorer precision of phosphorescence measurements, the method has not found as widespread use as has fluorometry.
• On the other hand, the potentially greater selectivity of phosphorescence procedures is attractive.
• Development of phosphorimetric methods that can be carried out at room temperature took two directions.
1. The first based upon the enhanced phosphorescence
that is observed for compounds adsorbed on solid surfaces, such as filter paper. In these applications, a solution of the analyte is dispersed on the solid, and the solvent is evaporated. The phosphorescence of the surface is then measured. Presumably the rigid matrix minimizes deactivation of the triplet state by external and internal conversions.
• The second is based on room-temperature method that involves solubilizing the analyte in detergent micelles in the presence of heavy metal ions.
Lifetime Measurements
• The measurement of luminescence lifetimes
was initially restricted to phosphorescent
systems, where decay times were long
enough to permit the easy measurement of
emitted intensity as a function of time.
• For analytical work, lifetime measurements
enhance the selectivity of luminescence
methods, because they permit the analysis of
mixtures containing two or more luminescent
species with different decay rates.
CHEMILUMINESCENCE
• The number of chemical reactions that produce chemiluminescence is small, thus limiting the procedure to a relatively small number of species.
• Nevertheless, some of the compounds that do react to give chemiluminescence are important components of the environment.
• Chemiluminescence is produced when a chemical reaction yields an electronically excited species, which emits light as it returns to its ground state.
• Chemiluminescence reactions are encountered in a number of biological systems, where the process is often termed bioluminescence.
• Examples of species that exhibit bioluminescence include the firefly, the sea pansy and certain jellyfish, bacteria, protozoa, and crustacea.
• Several relatively simple organic compounds also are capable of exhibiting chemiluminescence. The simplest type of reaction of such compounds to produce chemiluminescence can be formulated as
where C* represents the excited state of the
species C. Here, the luminescence spectrum
is that of the reaction product C
Measurement of Chemiluminescence
• The instrumentation may consist of only a suitable reaction vessel and a photomultiplier tube.
• Generally, no wavelength-restricting device is necessary, because the only source of radiation is the chemical reaction between the analyte and reagent.
• Several instrument manufacturers offer chemiluminescence photometers.
• The typical signal from a chemiluminescence experiment as a function of time rises rapidly to a maximum as mixing of reagent and analyte is complete; then more or less exponential decay of signal follows.
• Usually, the signal is integrated for a fixed period of time and compared with standards treated in an identical way.
• Often a linear relationship between signal and ,concentration is observed over a concentration range of several orders of magnitude.
spectral distribution of radiation emitted by the above reaction
A good example of chemiluminescence is the determination
of nitrogen monoxide:
NO + O3 NO2* + O2
NO2* NO2 + hv (= 600 to 2800 nm)
Analytical Applications of Chemiluminescence
• Chemiluminescence methods are generally
highly sensitive, because low light levels are
readily monitored in the absence of noise.
• Furthermore, radiation attenuation by a filter
or a monochromator is avoided.
• Detection limits are usually determined not
by detector sensitivity but rather by reagent
purity. They are in the ranges of ppb levels.
Analysis of Gases
Determination of nitrogen monoxide
• Ozone from an electrogenerator and the atmospheric
sample are drawn continuously into a reaction vessel
• Luminescence radiation is monitored by a
photomultiplier tube.
• A linear response is reported for nitrogen monoxide
concentrations of 1 ppb to 10,000 ppm.
• Instrumentally, for determination of nitrogen in solid or
liquid materials containing 0.1 to 30% nitrogen. The
samples are pyrolyzed in an oxygen atmosphere under
conditions whereby the nitrogen is converted
quantitatively to nitrogen monoxide; the latter is then
measured by the method just described.
Analysis of Inorganic Species in the Liquid Phase
• Many of the analyses carried out in the liquid phase
make use of organic chemiluminescing substances
containing the functional group
• These reagents react with oxygen, hydrogen peroxide, and many other strong oxidizing agents to produce a chemiluminescing oxidation product.
• Luminol is an example of these compounds. Its reaction with strong oxidants, such as oxygen, hydrogen peroxide, hypochlorite ion, and permanganate ion, in the presence of strong base is given below.
• Often a catalyst is required for this reaction to proceed at a useful rate.
• The emission produced matches the fluorescence spectrum of the product, 3-aminophthalate anion; the chemiluminescence appears blue and is centered around 425 nm.