describes the complete history, mechanisms, instrumentation(jablonski diagram), types, comparision and factors affecting, applications of fluorescence and phosphorescence and describes about quenching and stokes shift.

1. FLORESCENCE AND
PHOSPHORESCENCE
PRESENTED TO: SIR ATHAR
PRESENTED BY: SAMAWIA IQBAL
SAP I’D: 11007

2. CONTENTS
• INTRODUCTION
• BASIC MECHANISM
• ISTRUMENTATION
• PRINCIPLE
• TYPES
• DIFFERENCE BETWEEN THEM
• FACTORS AFFECTING
• APPLICATIONS
• REFERENCES
BACKGROUND
DEFINITION
JABLONSKI ENERGY DIAGRAM
QUENCHING
SINGLET AND TRIPLET STATES

3. INTRODUCTION
• LUMINISCENCE SPECTROSCOPY
luminescence spectroscopy deals with the study of the
emission
of radiations from a specie that has absorbed radiations. It has
three broad divisions.
FLORESCENCE PHOSPHORESCEN
CE
CHEMILUMINISCE
NCE

4. HISTO
RY
FLUORESCENCE :
An early observation of fluorescence was described in 1560 by Bernardino de
Sahagún and in 1565 by Nicolás Monardes in the infusion known as lignum
nephriticum (Latin for "kidney wood"). It was derived from the wood of two
tree species, Pterocarpus indicus and Eysenhardtia polystachya The chemical
compound responsible for this fluorescence is Matlaline, which is the
oxidation product of one of the flavonoids found In this wood.
PHOSPHORESCENCE :
It is derived from a Greek word “phosphor” meaning "which bears light". The
term phosphor has indeed been assigned since the Middle Ages to materials
that glow in the dark after exposure to light. There are many examples of
minerals reported a long time ago that exhibit this property, the most famous
of them (but not the first one) was the Bolognian phosphor discovered by a
cobbler of Bologna in 1602, Vincenzo Cascariolo.

5. Lignum nephriticum cup
made from the wood of the
tree, Pterocarpus indicus,
and a flask containing its
fluorescent solution.
Matlaline, the
fluorescent substance
in the wood of the
tree Eysenhardtia
polystachya.
Phosphorescent, europ
ium-
doped strontium silicat
e-aluminate oxide
powder under visible
light, long-wave UV
light, and in total
darkness.

6. DEFINITIONS
Fluorescence is the emission of light by a substance that has absorbed light or
other electromagnetic radiation. It is a form of luminescence. In most cases,
the emitted light has a longer wavelength, and therefore lower energy, than
the absorbed radiation. The most striking example of fluorescence occurs
when the absorbed radiation is in the ultraviolet region of the spectrum, and
thus invisible to the human eye, while the emitted light is in the visible
region, which gives the fluorescent substance a distinct color that can be seen
only when exposed to UV light.
Phosphorescence is a luminosity that is caused by the absorption of
radiation, in simple words it is a process in which energy absorbed by a
particular substance is released in the form of light.

7. MECHAN
ISMFluorescence occurs when an excited molecule, atom, or nanostructure,
relaxes to a lower energy state (possibly the ground state) through emission
of a photon. It may have been directly excited from the ground state S0 to
a singlet state S2 from ground state by the absorption of photon of energy
ɦνₑₓ and subsequently emits a photon of lower energy ɦνₑᵤ as it relaxes.
EXCITATION :
S0 + ɦνₑₓ S2
FLORESCENCE/ EMISSION :
S₂ S₁ + ɦνₑᵤ

8. PRINCIPL
ETHE PAULI EXCLUSION PRINCIPLE STATES
The Pauli Exclusion Principle states that, in an atom or
molecule, no two electrons can have the same four electronic
quantum numbers. As an orbital can contain a maximum of
only two electrons, the two electrons must have opposing
spins. This means if one is assigned an up-spin ( +1/2), the
other must be down-spin (-1/2).

9. SINGLET AND TRIPLET
STATES
• GROUND STATE :
Ground state, two electrons per orbital and have opposite spins.
• SINGLET EXCITED STATE :
Electrons in the higher energy orbital has the opposite spin
orientation relative to the electrons in lower orbital.
• TRIPLET EXCITED STATE:
The excited valence electron may spontaneously reverse its spin(
spin flip). This process is called intersystem crossing. Electrons in
both orbitals now have same spin orientation.

12. STOKES
SHIFT
The emitted light has a lower energy
(lower frequency, longer wavelength)
than the absorbed radiation; the
difference in these energies is known as
the Stokes shift.
“Stokes shift is the difference
(in energy, wavenumber or frequency
units) between positions of the band
maxima of the absorption and emission
spectra of the same electronic
transition”

13. QUENCHI
NG
 Fluorescence quenching refers to any process that decreases
the fluorescence intensity of a sample. A variety of molecular
interactions can result in quenching. These include excited-
state reactions, molecular rearrangements, energy transfer,
ground-state complex formation, and collisional quenching.
 Relaxation from an excited state can also occur through
transferring some or all of its energy to a second molecule
through an interaction known as fluorescence quenching.
Molecular oxygen (O2) is an extremely efficient quencher of
fluorescence just because of its unusual triplet ground state.

14. QUANTUM
YIELD

15. JABLONSKI DIAGRAM
In molecular spectroscopy, a Jablonski diagram is a diagram that
illustrates the electronic states of a molecule and the transitions
between them. The vibrational ground states of each electronic
state are indicated with thick lines, the higher vibrational states
with thinner lines.The diagram is named after the Polish
physicist Aleksander Jabłoński.
NON RADIOACTIVE DECAY
RADIOACTIVE DECAY

17. EXPLANATI
ON. several different electronic states exist (illustrated
as S(0), S(1), and S(2).
 Each electronic state is further subdivided into a number of
vibrational and rotational energy levels.
 The ground state for most organic molecules is an electronic
singlet in which all electrons are spin-paired (have opposite
spins).
 diagram illustrates the singlet ground (S(0)) state, as well as
the first (S(1)) and second (S(2)) excited singlet states as a
stack of horizontal lines.
 Absorption of light occurs very quickly (approximately a
femtosecond) in discrete amounts termed quanta and
corresponds to excitation of the fluorophore from the ground
state to an excited state.

18.  The absorption of a photon
of energy by a fluorophore,
which occurs due to an
interaction of the oscillating
electric field vector of the
light wave with charges
(electrons) in the molecule, is
an all or none phenomenon
and can only occur with
incident light of specific
wavelengths known
as absorption bands. If a
photon contains more energy
than is necessary for
transition, than the excess of
energy is converted to
vibrational and rotational
energy.
If a collision occurs
between a molecule and
photon having
insufficient energy to
promote a transition, no
absorption occurs.

19.  Immediately following absorption of a
photon, several processes will occur with
varying probabilities, but the most likely
will be relaxation to the lowest vibrational
energy level of the first excited state
(S(1) = 0). This process is known
as internal conversion or vibrational
relaxation (loss of energy in the absence
of light emission) and generally occurs in a
picosecond or less.
 An excited molecule exists in the lowest
excited singlet state (S(1)) for periods on
the order of nanoseconds (the longest
time period in the fluorescence process by
several orders of magnitude) before finally
relaxing to the ground state. If relaxation
from this long-lived state is accompanied
by emission of a photon, the process is
formally known as fluorescence.
The excited state can be
dissipated non radioactively,
as heat. The excited
fluorophore can collide with
an other molecule to transfer
energy in a second type of
non radioactive process,
quenching. Or a process
known as intersystem
crossing, to the lowest
excited triplet state with the
emission of photon, i.e.
phosphorescence or
transition back to the excited
singlet state that yields
delayed fluorescence.

20. Phosphorescence
decay is similar to
fluorescence, except
the electron undergoes
a spin conversion into a
"forbidden" triplet state
(T(1)) instead of the
lowest singlet excited
state, a process known
as intersystem
crossing.
PHOSPHORESC
ENCEEmission from the triplet state
occurs with lower energy relative
to fluorescence, hence emitted
photons have longer
wavelengths. With delayed
fluorescence, the electron first
decays into the triplet state, and
then crosses back over into the
lowest singlet excited state
before returning to the ground
state.

23. FLUORISCENCE
INSTRUMENTATION

24. While some fluorescence can be
detected with the eye, sophisticated
instruments have been built to detect
even the faintest fluorescence emitted
by a molecule.
The Filter fluorimeter
An older type of instrument for the
measurement of fluorescence spectra, and one
that is still used today, is the filter fluorimeter. It
consists of the following parts:
• an excitation source (like a lamp or laser),
• a primary filter,
• a sample chamber (also called a cuvette),
• a secondary filter, and
• a fluorescence detection system.
The filters only permit
radiation of certain
wavelengths (typically
the primary filter permits
short wavelengths
needed for excitation
and the secondary filter
permits long
wavelengths associated
with emission) and serve
to eliminate residual
radiation scatter. The
fluorescence detection
system consists of
photomultiplier tubes
(PMT) that amplify the
photon emission and
record and display the
signal electronically

25. Modern Fluorescence
Spectrophotometers
Most modern fluorescence spectrophotometers
are more advanced instruments than the filter
fluorimeter in that they can detect fluorescence
with higher precision and extraordinary
sensitivity. They are superior in wavelength
selectivity, flexibility, and convenience. A
Spectro fluorimeter is often equipped with the
following:
• a high-pressure xenon arc lamp,
• monochromators,
• a sample chamber (also called a
cuvette), and
• a fluorescence detection system.
The high-pressure xenon
arc lamp, used as the
excitation source, can
provide an energy
continuum that extends
from the ultraviolet into
the infrared.
monochromators which
allow for the production
of individual wavelengths
from a broad-band light
source. This makes it
possible for Spectro
fluorimeters to record
both excitation and
emission spectra.
monochromators allow
one to keep emission
fixed at a single
wavelength to obtain the
excitation spectrum it is
possible to keep
excitation fixed at a

26. Phosphorescence instrumentation

27. Instrumentation for molecular phosph
orescence must discriminate between
phosphorescence and fluorescence.
Since the lifetime
for fluorescence is much shorter than t
hat for phosphorescence, discriminatio
n is
easily achieved by incorporating a dela
y between exciting and measuring pho
sphorescent emission
 the two choppers are rotated out of phase, such
that fluorescent emission is blocked from the de
tector when the excitation source is focused on t
he sample, and the excitation source is blocked f
rom the sample when measuring the phosphore
scent emission.
phosphorescenc
e is such a slow p
rocess, provision
must be made to
prevent deactivat
ion of the excited
state by external
conversion
this has been done by
dissolving the sample
in a suitable solvent,
e.g. mixture of ethanol,
isopentane and diethyl
ether.

28. TYPES OF
FLUORESCENCEFluorescence can be divided into two groups on the basis of
 Wavelength of the emitted radiations
 Type of Phenomena
1- wavelength of emitted radiations
 Stokes fluorescence:
The wavelength of emitted radiation is longer than that
of the absorbed radiation.
 Anti stock’s fluorescence:
The wavelength of emitted radiation is shorter than the
absorbed radiation.

29.  Resonance fluorescence :
When the wavelength of emitted radiation is equal to
absorbed radiation(less observed type).
TYPES ON THE BASIS OF PHENOMENON:
 Prompt fluorescence
The release of electromagnetic energy is immediate or
from the singlet state.
 Delayed fluorescence
This results from two intersystem conversions, first
singlet-triplet and then triplet to ground state.

30. Delayed fluorescence:
It results from two intersystem crossing, first from singlet to
the triplet and then from triplet to the ground state.
 P-TYPE DELAYED FLUORESCENCE
 E-TYPE DELAYED FLUORISCENCE

31. FACTORS AFFECTING
FLUORESCENCE
• Conjugation
a molecule should posses conjugation(pi-electron) so that the visible and
ultra violet radiations could be absorbed.
• Nature of substituent groups
electron donating group can enhance fluorescence e.g. OH,NH2
Electron withdrawing groups decrease fluorescence e.g. COOH, NO2.
• Concentration
Fluorescence intensity is directly proportional to concentrations.
• Viscosity
Increased viscosity decreases the chances of collision of molecules
thereby decreasing fluorescence.

32. • Rigidity
more rigid the structure of molecule, more the intensity of fluorescence.
• Temperature
Increase in temperature leads to increase in the collision of molecules and
thus decreases the fluorescence intensity.
• Presence of oxygen
Presence of oxygen decreases the fluorescence thus de-aerated solutions
must be used.
• Atomic number
Atoms of higher atomic number decreases the chance of fluorescence and
increases the chance of phosphorescence.

35. APPLICATIONS
 LIGHTING:
• Common fluorescent lamps used for lighting.
 ANALYTICAL ANALYSIS:
• May analytical processes uses fluorescence to detect compounds from
HPLC flow.
 MICROSCOPY:
 Fluorescence in the life sciences is used generally as a non-destructive way
of tracking or analysis of biological molecules by means of the fluorescent
emission
 FLIM (Fluorescence Lifetime Imaging Microscopy) can be used to detect
certain bio-molecular interactions that manifest themselves by influencing
fluorescence lifetimes.
 FRET (Förster resonance energy transfer, also known as fluorescence
resonance energy transfer) is used to study protein interactions, detect
specific nucleic acid sequences and used as biosensors.

36.  FORENSIC:
Fingerprints can be visualized with fluorescent compounds such
as ninhydrin or DFO (1,8-Diazafluoren-9-one). Blood and other substances are
sometimes detected by fluorescent reagents, like fluorescein.
 NON-DESTRUCTIVE TESTING:
Fluorescent penetrant inspection is used to find cracks and other defects on
the surface of a part. Dye tracing, using fluorescent dyes, is used to find leaks
in liquid and gas plumbing systems.
 SIGNAGE:
Fluorescent colors are frequently used in signage, particularly road signs.
Fluorescent colors are generally recognizable at longer ranges than their non-
fluorescent counterparts, with fluorescent orange being particularly
noticeable.
 GLOW SHEETS:
"Glow Sheet" which used phosphorescent lines under writing paper to help
people write in low-light conditions.

37.  SHADOW WALL:
A shadow wall is created when a light flashes upon a person or object in front
of a phosphorescent screen which temporarily captures the shadow. The
screen or wall is painted with a glow-in-the-dark product that contains
phosphorescent compounds.
 DAILY USE ITEMS:
Everyday examples of phosphorescent materials are the glow-in-the-dark
toys, stickers, paint, wristwatch and clock dials that glow after being charged
with a bright light such as in any normal reading or room light.

38. REFERENCE
• https://www.alchemywebsite.com/bologna.htm
• https://en.wikipedia.org/wiki/Phosphorescence
• https://link.springer.com/chapter/10.1007%2F978-3-642-56853-
4_1
• https://en.wikipedia.org/wiki/Fluorescence
• https://www.slideshare.net/bijayauprety/fluorimetry-41214134
• https://chem.libretexts.org
• https://studylib.net/doc/7264211/molecular-luminescence-
spectrometry
• Molecular-Photoluminescence-Spectroscopy_29702
• https://micro.magnet.fsu.edu/primer/java/jablonski/lightandcolor
/index.html
• https://www.slideserve.com/laurence/chapter-15-molecular-
luminescence-spectrometry
• https://www.edinst.com/us/blog/jablonski-diagram/