Fluorescence and phosphorescence

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 byBernardino de
Sahagún and in 1565 byNicolás Monardes in theinfusionknown aslignum
nephriticum(Latinfor "kidney wood"). It was derived from the wood of two
tree species,Pterocarpus indicusandEysenhardtia polystachyaThe chemical
compound responsible for this fluorescence is Matlaline, which is the
oxidation product of one of theflavonoidsfound 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 phosphordiscovered by a
cobbler of Bologna in 1602, Vincenzo Cascariolo.

5. Lignum nephriticumcup
made from the wood of the
tree, Pterocarpus indicus,
and a flask containing its
fluorescentsolution.
Matlaline, the
fluorescent substance
in the wood of the
treeEysenhardtia
polystachya.
Phosphorescent,europ
ium-
dopedstrontiumsilicat
e-aluminate oxide
powder under visible
light, long-waveUV
light, and in total
darkness.

6. DEFINITIONS
Fluorescenceis the emission oflightby a substance that has absorbed light or
otherelectromagnetic radiation. It is a form ofluminescence.In most cases,
the emitted light has a longerwavelength, and therefore lower energy, than
the absorbed radiation.The most striking example of fluorescence occurs
when the absorbed radiation is in theultravioletregion of thespectrum, 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 toUV light.
Phosphorescenceis a luminositythat 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 oflight.

7. MECHAN
ISM
Fluorescence occurs when an excited molecule, atom, ornanostructure,
relaxes to a lower energy state (possibly theground state) through emission
of aphoton. It may have been directly excited from the ground state S
0to
asinglet state S
2from ground state by the absorption of photon of energy
ɦνₑₓ and subsequently emits a photon of lower energy ɦνₑᵤ as it relaxes.
EXCITATION :
S
0+ ɦνₑₓ S
2
FLORESCENCE/ EMISSION :
S₂ S
₁ + ɦνₑᵤ

8. PRINCIPL
E
THE PAULI EXCLUSION PRINCIPLE STATES
ThePauliExclusionPrinciplestatesthat,inanatomor
molecule,notwoelectronscanhavethesamefourelectronic
quantumnumbers.Asanorbitalcancontainamaximumof
onlytwoelectrons,thetwoelectronsmusthaveopposing
spins.Thismeansifoneisassignedanup-spin(+1/2),the
othermustbedown-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
theStokes shift.
“Stokes shiftis the difference
(inenergy,wavenumberorfrequency
units) between positions of the band
maxima of theabsorptionandemission
spectra of the same electronic
transition”

13. QUENCHI
NG
Fluorescence quenchingrefers to anyprocessthat decreases
thefluorescenceintensity of a sample. A variety of molecular
interactions can result inquenching. These include excited-
state reactions, molecular rearrangements, energy transfer,
ground-state complex formation, and collisionalquenching.
Relaxation from an excited state can also occur through
transferring some or all of its energy to a second molecule
through an interaction known asfluorescence quenching.
Molecularoxygen(O
2) is an extremely efficient quencher of
fluorescence just because of its unusual triplet ground state.

14. QUANTUM
YIELD

15. JABLONSKI DIAGRAM
In molecularspectroscopy, aJablonski diagramis a diagram that
illustrates theelectronic statesof amoleculeand 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
physicistAleksander Jabłoński.
NON RADIOACTIVE DECAY
RADIOACTIVE DECAY

17. EXPLANATI
ON.several different electronic states exist (illustrated
asS(0),S(1), andS(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 termedquantaand
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
asabsorption bands. If a
photon contains more energy
than is necessary for
transition, than the excess of
energy is converted to
vibrationaland 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
asinternal conversionorvibrational
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 stateis 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.
phosphorescenceor
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
asintersystem
crossing.
PHOSPHORESC
ENCE
Emissionfrom 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
Mostmodernfluorescencespectrophotometers
aremoreadvancedinstrumentsthanthefilter
fluorimeterinthattheycandetectfluorescence
withhigherprecisionandextraordinary
sensitivity.Theyaresuperiorinwavelength
selectivity,flexibility,andconvenience.A
Spectrofluorimeterisoftenequippedwiththe
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.
monochromatorswhich
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
single wavelength, to

26. Phosphorescence instrumentation

27. Instrumentationformolecularphosph
orescencemust discriminate between
phosphorescence and fluorescence.
Since thelifetime
forfluorescenceismuchshorterthant
hatforphosphorescence,discriminatio
nis
easilyachievedbyincorporatingadela
ybetweenexcitingandmeasuringpho
sphorescentemission
thetwochoppersarerotatedoutofphase,such
thatfluorescentemissionisblockedfromthede
tectorwhentheexcitationsourceisfocusedont
hesample,andtheexcitationsourceisblockedf
romthesamplewhenmeasuringthephosphore
scentemission.
phosphorescenc
eissuchaslowp
rocess,provision
mustbemadeto
preventdeactivat
ionoftheexcited
statebyexternal
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
FLUORESCENCE
Fluorescence 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
HPLCflow.
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 asfluorescence
resonance energy transfer) is used to study protein interactions, detect
specific nucleic acid sequences and used as biosensors.

36. FORENSIC:
Fingerprintscan be visualized with fluorescent compounds such
asninhydrinor DFO (1,8-Diazafluoren-9-one). Blood and other substances are
sometimes detected by fluorescent reagents, likefluorescein.
NON-DESTRUCTIVE TESTING:
Fluorescent penetrant inspectionis 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 insignage, 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 productthat contains
phosphorescent compounds.
DAILY USE ITEMS:
Everyday examples ofphosphorescentmaterials 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/