Molecular Fluorescence

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Molecular Fluorescence

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Title: Molecular Fluorescence

1
Chapter 9
Molecular Fluorescence and Phosphorescence
2
Sources of Luminescence

Luminescence can be classifieds according to the
source of excitation into
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

3
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. Chemilumin
escence is based on an excited species formed by
a chemical reaction.
4

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.

5
Excitation and de-excitation process
6
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)

8
Spin Orientations
9

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 cant happen

10
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).
11
Difference between triplet and singlet states

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

12
Energy of a Molecule (Jablonski energy-level
diagram)
13
Energy Levels for Luminescence Transitions
quenching
14
Fluorescence in the Jablonski energy-level
diagram
15
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.

17
Deactivation Processes

The molecule can rapidly dissipate excess
vibrational energy as
heat by collision with solvent molecules through
vibrational relaxation process
2. EMR

Internal Conversion IC
Inter System Crossing ISC
- Quenching
- Fluorescence
- Phosphorescence

18
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.

20
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.

21
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

24
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.

25
Intersystem crossing

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.

26
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 excited state
energy
v0
Electronic ground state
v0
27
Fluorescence
Most of the fluorescence spectrum is shifted to
lower energies (longer wavelengths), compared to
the absorption spectrum.
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 excited state
energy
v0
Electronic ground state
v0
28
Mirror Image Spectra
The above spectra are plotted as amplitude versus
wave number. When plotted versus wavelength
the mirror effect is not as pronounced.
29

The shortest wavelength in the fluorescence
spectrum is the longest wavelength in the
absorption spectrum

30
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.

31
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 v0 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 excited state
energy
Electronic ground state
32
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 excited state
energy
Electronic ground state
33
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.
34
Quenching

Energy gets transferred to the quencher, usually
through collisions with a nearby residue or
molecule
This reduces photon emissions and decreases
fluorescence intensity.

35
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.

36
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
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
37
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

common quenchers are oxygen, molecules with heavy
atoms, and molecules with unpaired spins
38
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
39

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 kfS1
?f (kIC kISC kf kQ
Q) S1
?f kf / (kIC kISC kf kQ Q)

40
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 (?) 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
fp/ff 0.093 0.068
5.2 16.4 gt1000

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 kVR kQP Qp
and ?p / ?t ?P / ?PR
Kp First order decay const of T1 to S0 state
kVR 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

45
Effect of Concentration on Fluorescent Intensity
If ?I ?f ?f (Io IT) .(1) IT Io X 10
-?bc (2) Where ? is the molar absorptivity
of the fluorescing molecule. Substituting Eq 2
in Eeq 1 If ?fIo (1 10 -?bc ) . (3) The
exponential term in Eq 3 can be expanded as a
Maclaurin series to If ?fIo 2.303 ?bc -
(2.303 ?bc )2/2! (2.303 ?bc )3/3!.. ) Provided
2.303 ?bc lt 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 ?bc Or
If kC. If VS C is straight line at low
concentration
46
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47
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48

Factors responsible for non linearity
The concentration When 2.303 ?bc is more than
0.05, the linearity is lost
Self quenching collisions between excited
molecules
Self absorption When the wave length of emission
overlaps an absorption peak. Fluorescence is then
decreased as the emitted beam traverse the
solution

49
Excitation and Emission Spectra

Fluorescing molecules are characterized by two
types
of spectra
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

50
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
51

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)

52
Fluorescent Excitation and Emission Spectra
53
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
54
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.
55
Electronic Transition Types in Fluorescence

Seldom to have fluorescence by absorbing Uv at lt
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.

56
Quantum Efficiency and Transition Type

?f (? - ?) gt ?f (? - n) transition
? 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.

59
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

61
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.

63
Fluorescence and Structure
64
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.

65
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.

68
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.

69
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.

70
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

71
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72
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.

73
Fluorescence and Phosphorescence Instruments
74

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

75
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

76
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.

77
Filters And Monochromators

Both interference and absorption filters have
been employed in fluorometers.
Most spectrofluorometers are equipped with
grating monochromators.

78
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

79
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.

80
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.

81
Nearly all fluorometers (spectrofluorometers)
are double-beam systems.
82
Spectrofluorometer
83
Fluorometer or Spectrofluorometer
84
Filter Fluorometer
85
Spectrofluorometers

spectrofluoters 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

86

Spectrofluorometer based on Array Transducers
Transducer is a two-dimensional device that sees
the excitation and emission radiation in two
planes
Observe Fluorescent Excitation and Emission
Spectra Simultaneously
87
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
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).

89
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 Detecor so that only
fluorescence is observed.
90
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.

91
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.

92
Cations that form Fluorescing Chelates

Two factors greatly limit the number of
transition-metal ions that
form fluorescing chelates.
Many of these ions are paramagnetic this
property increases the rate of intersystem
crossing to the triplet state. Deactivation by
fluorescence is thus unlikely, although
phosphorescence may be observed.
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.

93
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.

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95
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.

96
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.
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.

98
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.

99
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100
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

101
where C represents the excited state of the
species C. Here, the luminescence spectrum is
that of the reaction product C
102
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 max

103

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.

104
A good example of chemiluminescence is the
determination of nitric oxide NO O3
NO2 O2 NO2
NO2 hv (? 600 to 2800 nm)
spectral distribution of radiation emitted by the
above reaction
105
Analytical Applications of Chemiluminescence

Chemiluminescence methods are generally highly
sensitive, because low light levels are readily
monitored 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.

106
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.

107
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

108

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.