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.