Fluorescence

Spectroscopy
Presented by;

GUL MUHAMMAD and ALMAS

KANWAL
[Link](Pharma . Chem)
Spectroscopy:
“Study of spectrum, to identify
substances”
Types of spectra:
(a) Continuous spectra.
(b) Absorption spectra.
(c) Emission spectra.
 Emission
Process:
High energy level

emission
Energy
Absorb energy

Electrons ground level

Electronic state
Transitions:
Jablonski Diagram
Transition
possibilities:
 Fluorescence
 Phosphorescence
 Collision
deactivation
Fluorescence
Spectroscopy:
FLUORESCENC
E:
Fluorescence is the emission
of light by a substance that
has absorbed light or other
electromagnetic radiation of
a different wavelength
 Fluorescence
spectroscopy??
Principle of Fluorescence
Fluorescence Spectroscopy
occurs when a molecule
absorbs photons from the u.v-visible
light spectrum (200-800nm), causing
transition to a high-energy electronic
state and then emits photons as it
returns to its initial state, in less than
10-9 sec. Some energy within the
molecule, is lost through heat or
vibration so that emitted energy is
less than the exciting energy; i.e., the
emission wavelength is always longer
than the excitation wavelength.
 The absorption of a photon of energy, or
wavelength, can lead to re-emission of one or
more photons at lower energies, or longer
wavelengths
Fluorescence related
Terminologies:
Fluorescent dye (Fluorophore)
Excitation
Extinction coefficient
Emission
Quantum yield
Stokes shift
Spectrum
Fluorophore –The λ and time resolution req
by instrument is det by its spectral
properties
Excitation –Process of absorbing light energy
Extinction coefficient-Efficiency of
absorbing light energy ;it’s value is
wavelength dependent
Emission-Process of releasing light energy
Quantum yield-Efficiency of releasing energy
generally in form of fluorescent light ;not
wavelength dependent
Stokes shift-Difference in energy b/w
excitation and emission.
Spectrum-Distribution of excitation and
Factors affecting
fluorescence intensity:
 Nature of molecule
 Effect of Concentration
 Nature of substituent
group
 Rigidity of structure
 Temperature/Viscosity
 Effect of pH
 Oxygen
 Quenching
01:Nature of molecule:

Fluorescence is most commonly observed

in compounds containing aromatic
functional group.

Molecule must have unsaturation (i.e.

conjugation), So that UV-Visible radiation
can be absorbed.
02:Effect of Concentration
If more number of molecules present ,
They absorb more radiation and So emit
more radiation.

Relation of concentration &

Fluorescence.

FαC
03:Nature of substituent
group:
Normally unsubstituted aromatic
hydrocarbons show fluorescence

Electron donating group like amino (-NH 2

), Hydroxy (-OH) groups enhance
fluorescence intensity

Electron withdrawing groups like Nitro

(NO 2 ) , Carboxylic (-COOH) group reduce
fluorescence intensity

Groups like SO3H or NH4+ have no effect

on fluorescence intensity.
04:Rigidity of structures:

Rigid structure More

fluorescence intensity
([Link])

Flexible structure Less

fluorescence intensity
([Link]).
05:Temperature/
Viscosity
effects:
Temperature affect the
viscosity of the medium and
number of collisions of
fluorophore with solvent
molecules results in decrease in
Fluorescence intensity.
06:Effect of pH:

This depends on chemical structure

of molecules.

E.g; Phenol in acidic condition are

undissociated & don’t give
fluorescence. In alkaline condition
they are dissociated & give good
fluorescent.

Resolved; particular buffer solutions

are recommended.
07:Oxygen:
Interfere in two ways

01: By direct oxidation.

02: By quenching effect.

09:Quenchin
g
Decrease of fluorescence intensity
by interaction of the excited state
of the fluorophore with its
surroundings is known as
quenching

Example; Quinine fluorescence is

quenched by the presence of
halide ion.
nstrumentation:
The basic instrument is a
spectrofluorometer

COMPONENTS:

 light source
 excitation monochromator (primary
filter)
 a sample cell
 emission monochromator (secondary
filter)
 a detector
 and a read out device.
Light source:

1. XENON ARC LAMP

2. MERCURY ARC LAMP
3. LIGHT EMITTING DIODES (LED)
01: Xenon arc lamp
Spectrum is continuous
over the range between
over 250-600 nm

02: Mercury lamp

Produce intense
spectrum above 350nm

03: Light emitting

diodes (LED)
(wave length from 350
nm to 1300nm)
Monochromato
r
1:excitation
monochromator:
Isolate absorbed radiations.

2:emission
monochromator :
Isolate radiation emitted

Example: Prism,
Grating or
Filter
Sample holder
(fluorescent cuvette)

All surface of sample

holder are polished

Example: Quartz,
Optical glass,
Plastic
cells
Detector:
photomultiplier tube
S5 TYPE .
S20 TYPE
photo diode array

Readout
Device:
meter or
digital
display
Microprocessor
 Guiding Principles for
Imaging Fluorescence:
Select proper dye for the job
Optimize light conditions (Proper light
source,filter,detectors)
Minimize excitation exposure
(minimize bleaching, reduce sample
contradictions)
Maximize dynamic range (contrast
depend on this)
Minimize background signal
(maximize sensitivity)
 Applications of
Fluorescence
spectroscopy:
Extremely sensitive method of
detecting and observing
molecules
Can detect femta molar (10-15)
quantities
Can detect single molecules
Time scale –
microsecond,nanosecond
Observe motion in proteins and
bio-molecules.
Medical Applications of
Fluorescence
spectroscopy:
Spectral properties of fluorescent
probes in the life sciences finds
additional applications
in:Microscopy,Immunology,Biotechn
ology,Forensics etc
Fluorometric techniques are now a
days employed to determine
conc./levels of 5-
hydroxyindoleacetic acid and
protein in the cerebrospinal fluid
Medical Applications of
Fluorescence
spectroscopy:
Endoscopic examination of oral
and oesophageal cancers
Characterization of new
formulations in-vivo
In-vitro study of new drugs
Optimize procedures
Click icon to add picture

Endothelial cells under the

microscope
Click icon to add picture
 Experimental Assays
That utilize
Fluorescence:
Environmental monitoring
DNA sequencing
Fluorescence in-situ
hybridization(FISH)
ELISA (Enzyme linked immuno-sorbent
assay)
FLIM (fluorescence Lifetime Imaging
Microscopy)
Cell and molecular biology
FRET (Fluorescence resonance energy
 Compounds which
exhibit Fluorescence:
Aromatic ring with –OH,-OC and
alkyl amine groups
Heterocyclic ring
Highly conjugated structure
Compounds which show π -π*
transition during excitation
process shows strong properties
of fluorescence. For example
benzene
Phenol & phenolic compounds
 Latest Advancement in
Fluorescence
Spectroscopy:
I. Time-resolved fluorescence
spectroscopy
It provides fluorescence intensity decay
in terms of lifetimes (femto-second
time)
Advantages:
 Enhance the discrimination among
fluorophores (overlapping emission spectra)
 Sensitive to various parameters of the
biological microenvironment means it
facilitates medical research e.g TRFS data
correlates with pathological features and
Time-resolved fluorescence
spectroscopy
Time-resolved laser-induced fluorescence
spectroscopy (tr-LIFS)
 II. Atomic-Fluorescence
Spectroscopy (AFS)
Atomic fluorescence is
the optical emission
from gas-phase atoms
that have been excited
to higher energy Levels
by absorption of
electromagnetic radiation
 Atomic-Fluorescence
Spectroscopy (AFS)
Advantages:
fluorescence detection has the
greater sensitivity achievable
because the fluorescence signal has a
very low background.
AFS Analytical applications include
flames and plasmas diagnostics, and
enhanced sensitivity in atomic
(atom’s struc) analysis.
 III. Advanced Fluorescence
Technology (AFT)
AFT - How It Works??
AFT is the next generation of intense multi-
application pulsed light technology . It
converts unused short-wavelength UV light
into the usable spectrum by each application
through a special filtering system. It
increases emission and penetration, for
effective treatments.
AFT also delivers Equally Distributed
Fluence, which means that every pulse has
uniform energy across the entire [Link]
yields a more efficient system for ideal
clinical improvements, less discomfort ,more
safety and minimal skin damage.
Advanced Fluorescence
Technology (AFT)
 Advanced Fluorescence
Technology (AFT)
How does AFT treat Sun Damage??
FDA-approved AFT removing the sun
damage. The blue light penetrates
just deep enough into the tissue to
reach the target without adversely
affecting the surrounding skin.
What else can AFT treat??
Age spots,Fine and coarse wrinkles,
Freckles,Rosacea,Vascular
lesions,Swelling and treatment of
active acne.
AFT
Advanced Fluorescence
Technology (AFT)
[Link] Analyzer

A reliable solution for research

and routine,used for multi-
purpose analysis, its
Accurate,Flexibile and Easy to
Use
With its open and robust design,
Haematology Analyser may run
unique fluid types, such as
broncho-alveolar lavage fluid
and bone marrow, with ease.
Haematology Analyzer
 [Link] fluorometer

The Qubit fluorometer is a small

instrument used for
quantification of DNA, RNA, and
protein and used in many
different [Link]
fluorometer uses fluorescent
dyes to determine the
concentration of nucleic acids
and proteins in a sample.
Qubit fluorometer