1

LUMINESCENCE

FLUORESCENCE
PHOSPHORESCENCE
CH2

H N Quinine
HO
H
O
H3C

N
 2

OBJECTIVES

By the end of this session, the student should be able to:

1. Define luminescence, fluorescence and phosphorescence.
2. Explain how luminescence happens
3. Distinguish absorbance, excitation and emission spectra
4. Describe the set-up and the components of a fluorometer
5. Recognize molecular features causing fluorescence
6. Define parameters that influence molecular fluorescence
7. State applications of fluorescence spectroscopy
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HISTORY
 4

WHAT IS MOLECULAR LUMINESCENCE ?

➢ Chemiluminescence excitation resulting from a

chemical reaction
➢ Phosphorescence excitation by
➢ Molecular Fluorescence absorption of photons:
PHOTOLUMINESCENCE
➢ BASIC PRINCIPLE:
1st: molecules are excited (outer shell electrons like in
absorbance phenomenon)
2nd: excited species give an emission spectrum that
provides information for quantitation and
qualification
Fluorescence is short-lived, with luminescence ceasing
almost immediately (<10-5 sec) ,while phosphorescence
features luminescence from 10-4 to several seconds.
 5
Why Use ABSORBANCE MEASUREMENT
Molecular Changing transmittance from 100% to 99% is
Luminescence ? hard to measure such a small absorbance
because the background is so bright.
➢WHAT MAKES THESE
TECHNIQUES INTERESTING: T=P/Po=0.99
A= -log T= 0.004
▪sensitivity: ppb range (3 !!!!!!!!!!!!!!
orders of magnitudes higher
compared to absorption
spectroscopy).
▪large linear concentration
ranges
➢LIMITING FACTORS:
▪enormous sensitivity implies
FLUORESCENCE MEASUREMENT
purity of analytes (or prior Observing fluorescence of 1% of
purification) molecules
▪only few molecules exhibit
luminescence, compared to
those that absorb UV or visible
electromagnetic radiation
 6

SINGLET AND TRIPLET STATES

PAULI EXCLUSION PRINCIPLE: “no
two electrons in an atom can have
excited the same set of 4 quantum
singlet state numbers”, in other words:
two electrons in the same orbital
Excited triplet state is of less
must have opposite spins (we say:
energy than excited singlet they are "paired": no net magnetic
state. field = the molecule will be
Singlet to triplet transitions are "diamagnetic"), molecule with
far less probable than unpaired electrons (e.g. triplet state)
singlet/singlet transitions.
possess magnetic moment, are
excited attracted by magnetic field, are
called "paramagnetic"
triplet state

Physicochemical properties of
molecules in triplet state can differ
ground state significantly from those of singlet
state molecules.
 7

ELECTRONIC AND VIBRATIONAL LEVELS

➢ S0: ground state of a molecule at ambient temperature,
all of the molecules in a solution
➢ S1 and S2: excited singlet states

➢ T1: lowest energetic triplet state, usually of less energy

than lowest energetic excited singlet state S1. Same E
S1
Each of these states features various
vibrational levels – this permits energetic T1
similarity (and even equivalence) of
different electronic spin states of a molecule.

Because Singlet / triplet transitions are less probable than singlet /

singlet transition (because spin conversion is necessary) ,
thus the average lifetime of an excited triplet state is 10-4 sec and
more, while excited singlet state lifetime is 10-8 to 10-5 s.
 8

DEACTIVATION PROCESS
An excited molecule can return to its ground state by a combination of
several mechanistic steps:
•Two of these steps, fluorescence and phosphorescence, involve the
emission of a photon of radiation.
•The other deactivation steps are radiationless processes.
What kind of deactivation does occur?
The favored route to the ground state is always the one that
minimizes the lifetime of the excited state.
Thus, if deactivation by fluorescence is rapid with respect to
radiationless processes, such emission is observed.
If a radiationless path has a more favorable rate constant,
fluorescence is either absent or less intense.
Photoluminescence is limited to a small number of systems
incorporating structural and environmental features that cause
that the rate of radiationless deactivation be slowed in favor of
emission of photons.
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ELECTRON TRANSITIONS
•Photon absorption rate:
S2 10-14 to 10- 15 s !!
S1 •Fluorescence emission rate is
directly related to molar
T1 absorptivity.   103 to 105:
lifetime of excited state
10-7 to 10-9s; weakly absorbing

Low E, Long λ
systems: lifetime 10-6 to 10-5 s.
High E, Short λ

•External conversion involves

energy transfer between the excited
molecule and the solvent or other
solutes (collisional QUENCHING)
•Dissociation and pre-
S0
Energy

dissociation: rupture of chemical

λ1
absorption
Internal conversion
energetically. when 2 levels
are sufficiently close
bond due to high energy of
vibrational vibrational state
levels
λ2 λ3 intersystem crossing
(reversal of spin), common in molecules
Fluorescence always
containing heavy atoms or when
from lowest vibrational level of paramagnetic species are present (O in
2
an excited electronic state solution) → fluorescence is decreased.
phosphorescence vibrational relaxation due to
collisions between the molecules of the
excited species and those of the solvent
 10

VIBRATIONAL RELAXATION I
➢ 1st Observation :
Upon excitation different vibrational levels can be achieved,
in solution any excess vibrational energy is lost as
consequence of collisions between the molecules of the
excited species and solvent molecules
▪ Result:
Energy transfer to solvent and minuscule warming,
lifetime of vibrationally excited species: 10-12 sec and less.
▪ Consequence:
Fluorescence (and Phosphorescence) of an analyte in
solution always occurs due to electron transition
from a vibrational ground state.
Fluorescence due to any chosen electron transition (from any vibrational
ground state) produces less energy than the previous excitation had
absorbed. → Fluorescence signals of a molecule (moiety) generally appear at
longer wavelengths than the corresponding absorbance signals (STOKES shift).
 11

VIBRATIONAL RELAXATION II
➢2nd Observation:
Upon luminescence different vibrational levels can be achieved, in
solution any excess vibrational energy is lost as consequence of
collisions between the molecules of the excited species and solvent
molecules

▪Consequence I: fluorescence (and

phosphorescence) of an analyte do
not give sharp signals but diffuse
bands.

▪Consequence II: The fluorescence

spectrum of an analyte often is more
or less similar to its absorbance
spectrum.
 12

FRANCK-CONDON-PRINCIPLE
Electron transitions are so fast (10-15 s) that each atom has nearly the
same position before and after a transition; this is due to the small
mass of the electrons (FRANCK-CONDON principle).
Thus, molecular geometry initially is kept, and higher vibrational levels
are achieved upon absorption. Upon relaxation to ground state, the
same happens: molecular geometry is initially kept, so the vibrational
level that is reached first is NOT the ground state.
 13

ABSORBANCE, EXCITATION & EMISSION SPECTRA

Emission spectra are measured by holding excitation radiation fixed ( ex ) at one

particular wavelength and scanning through the emitted radiation. Graph:
emission intensity vs. emission wavelength.
Excitation spectra are measured by varying excitation wavelength and measuring
emitted light at one particular wavelength ( em): Graph: excitation intensity vs.
excitation wavelength. Because the 1st step in generating fluorescence is absorption
of radiation to create excited states, an excitation spectrum is essentially
identical to an absorbance spectrum taken under the same conditions.
 14

PHOSPHORESCENCE
After intersystem crossing from singlet to triplet state,
deactivation can occur by internal or external conversion or by
phosphorescence.
Since triplet-to-singlet conversions are comparatively
improbable events, the average lifetime of an excited triplet
state is 10-4 to 10 sec and more.
Thus, emission from such transition may persist for some time
after irradiation has been discontinued.
The other deactivation
transitions compete strongly T1
with phosphorescence, so
this phenomenon is usually
observed at low
S0
temperatures, in highly
viscous media or at
molecules being adsorbed on
surfaces.
 15

THE SHAPE OF LUMINESCENCE SPECTRA

I. Phosphorescence and Fluorescence (emission) Spectrum
both come at longer wavelengths compared to absorbance
spectrum of the same molecule (Stokes shift).

II. Phosphorescence
comes at lower energy =
at longer wavelengths
than fluorescence from
the same molecule.

III. Fluorescence (emission) Spectrum of a molecule is

more or less similar to its absorbance spectrum.
IV.  max of a Fluorescence (emission) Spectrum is slightly
shifted to longer wavelengths compared to  max of the
absorbance spectrum (Frank-Condon principle) because
the luminiscence process involves the electronic energy
change minus a vibrational energy change.
 16

What Do We Make Use Of?

I0 (radiant intensity) I (transmitted intensity)

© Dr. Rasha Hanafi, Lecture 4, Luminescence, 03-10-
GUC 2012
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Measurement: Fluorometers

Filter Fluorometers:
•rather simple and cheap!
•employ absorption or interference filters
for limiting  ex and  em
Spectrofluorometers:
• more sophisticated and expensive!
•allow generation of both excitation and
emission spectra,
• employ two grating monochromators
 18

MEASUREMENT: FLUOROMETERS (CONTD.)

SPECTROFLUOROMETER

A fluorescence signal produced when “a cell

or particle labeled with a fluorochrome”
passes through a laser excitation beam.
centered while they pass through the laser
beam
 19

LIGHT SOURCES OF FLUOROMETERS

➢ in Spectrofluorimeter:
continuous radiation required
i) 75- to 450W high pressure xenon arc lamp, emitting 300
to 1300 nm
large power supply needed (5 to 20 A at 15 to 30 V)
ii) tunable dye LASERs – comparatively expensive;
advantages: suitable for small samples (L or less), if highly
monochromatic excitation is required, or for remote sensing
 20

OTHER PARTS OF FLUOROMETERS

➢ excitation and emission
monochromator:
▪ interference and absorption filters for filter
fluorometers

▪ grating monochromators for spectrofluorimeter

➢ sample cell:
▪ cylindrical and rectangular cells of glass or quartz
▪ any fingerprints are even more disturbing than
in absorbance spectroscopy
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OTHER PARTS OF FLUOROMETERS

➢ detector:
▪ the most common transducers are photomultiplier tubes (PMT)
run in photon counting mode
▪ the final detector output (fluorescence signal) is the ratio (division!)
between the sample beam’s PMT signal intensity and the reference
beam’s PMT signal

photon counting mode (applied

for low intensity radiation):
analog signal is converted to a train
of digital pulses → radiant power is
proportional to the number of pulses
per unit time.
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PRINCIPLE
“Quantum yield” or “quantum efficiency”:
ΦF= ratio of numbers of molecules luminescent to the total
number of excited molecules(photons absorbed).

kf kf
Q= =
k k f + ki + kec + kic + k pd + k d
Transition types:
normally,  ex for fluorescence is >250 nm.
radiation of lower  is too energetic and may cause dissociation
or pre-dissociation of the molecules in excited states, thus their
deactivation.
As a consequence, either *→ n or *→  transition are
responsible for fluorescence, and not * → .
 CONCENTRATION AND
FLUORESCENCE INTENSITY
• The power of fluorescent radiation, F, is
proportional to the radiant power of the
excitation beam absorbed by the species able to
undergo fluorescence:
F = ΦF(P0 - P)
where P0 is the power incident on the sample, P
is the power after it traverses a length b of the
solution and ΦF is a constant which depends
upon experimental factors and the quantum
efficiency of fluorescence.
 CONCENTRATION AND
FLUORESCENCE INTENSITY

• Beer's law can be rearranged to give:

P/P0 = 10-bc
where A = bc is the absorbance.
Substitution gives:
F = ΦF P0(1 - 10- bc)
• This is the fluorescence law
• Unlike Beer’s Law fluorescence isn’t in
general linear with concentration.
 CONCENTRATION AND
FLUORESCENCE INTENSITY
• This expression can be expanded (Taylor series):
 (2.3bc) 2 (2.3bc) 3 
F =  P0 2.3bc - + − 
 2! 3! 

• To a good approximation if bc is small (< 0.05) the

higher-order terms are nearly zero, we have:

F = 2.3K'bcP0
At higher concentration
F= P0 ΦF
 CONCENTRATION AND
FLUORESCENCE INTENSITY
which demonstrates two important points:

• that at low concentrations fluorescence

intensity is proportional to concentration;

• that fluorescence is proportional to the

incident power in the incident radiation at
the absorption frequency.
 CONCENTRATION AND
FLUORESCENCE INTENSITY

For a
concentration
above c1 the
calibration
curve is no
c1 longer linear.

Conc. of fluorescing species

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FLUORESCENCE SPECTRA

CH2

H N

HO
H
O
H3C

Quinine (1R,3R,4S,8S,9R)
 29

SUMMARY
1. When a molecule absorbs light, it is promoted to an excited state from
which it may return to the ground state by radiationless processes or by
FLUORESCENCE (singlet→ singlet) or PHOSPHORESCNECE (triplet →
singlet emission).
2. Any form of luminescence is potentially useful in QUANTITATIVE
ANALYSIS, because emission intensity is proportional to sample
concentration at low concentration.
3. An EXCITATION spectrum (a graph of excitation intensity versus excitation
wavelength) is similar to an absorption spectrum (a graph of absorbance
versus wavelength). An emission spectrum is observed at lower energy
than the absorption spectrum due to Stokes shift and Frank Condon
Principle.
4. A molecule that is not fluorescent can be analyzed by attaching a
fluorescent group to it (FLUOROPHOR).
5. Light emitted from a chemical reaction (CHEMILUMINESCENCE) can also
be used for quantitative analysis.
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FACTORS AFFECTING FLUORESCENCE
❑ Molecules that are aromatic or contain multiple conjugated double bonds with a
high degree of resonance stability.

❑ Substituent's that strongly affects fluorescence are—NH2 ,-OH, -F, OCH3 ,-

NHCH3 (enhance the fluorescence) and electron withdrawing group –Cl, -Br,-I,
NHCOCH3 , -NO2 , -COOH (decrease the fluorescence).

❑ Molecular rigidity reduces the internal conversion, hence more fluorescence for
e.g. fluorescein and eosin are strongly fluorescent but phenolphthalein which is
nonrigid does not fluorescent.

❑ pH affects fluorescence, both phenol and anisole fluoresce at pH 7, but at pH 12

phenol is converted to nonfluorescent anion, whereas anisole remain unchanged.

❑ The formation of chelates with metal ions also promotes fluorescence by

promoting rigidity. The introduction of paramagnetic metal ions, such as copper
and nickel gives rise to phosphorescence but not fluorescence.
❑ Dissolved oxygen is a strong quencher of fluorescence, since oxygen is
paramagnetic. Oxygen must be removed by degassing.
 APPLICATIONS

A. Determination of polyaromatic
hydrocarbons

– Benzo[a]pyrene is a product of incomplete

combustion and found in coal tar.
 APPLICATIONS
• Benzo[a]pyrene, is a
5-ring polycyclic
aromatic hydrocarbon
that is mutagenic and
highly carcinogenic

• It is found in tobacco
smoke and tar

• The epoxide of this

molecule intercalates
in DNA, covalently
bonding to the guanine
base nucleotide
 APPLICATIONS
Excitation and
fluorescence spectra for
benzo(a)pyrene in
H2SO4. In the diagram
the solid line is the Benzo(a)pyrene
excitation spectrum (the
fluorescence signal is
measured at 545 nm as
the exciting wavelength
is varied). The dashed
line is the fluorescence
spectrum (the exciting
wavelength is fixed at
520 nm while the
wavelength of collected
fluorescence is varied).
 APPLICATIONS

B. Fluorimetric Drug
Analysis
• Many drugs
possess high
quantum efficiency
for fluorescence.
For example,
quinine can be
Quinine
detected at levels
below 1 ppb.
 APPLICATIONS
• In addition to
ethical drugs such
as quinine, many
drugs of abuse
fluoresce directly.
For example
lysergic acid
diethylamide (LSD)
whose structure is:
 APPLICATIONS
• Because LSD is active in minute quantities (as little as
50 g taken orally) an extremely sensitive methods of
analysis is required. Fluorimetricaly LSD is usually
determined in urine from a sample of about 5mL in
volume. The sample is made alkaline and the LSD is
extracted into an organic phase consisting of n-heptane
and amyl alcohol. This is a "clean-up" procedure that
removes potential interference and increases sensitivity.
The LSD is then back-extracted into an acid solution and
measured directly using and excitation wavelength of
335 nm and a fluorescence wavelength of 435 nm. The
limit of detection is approximately 1 ppb: