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2. Fluorescence and phosphorescence
• Fluorescence and phosphorescence are the deactivation processes
by which a molecule gives out the absorbed energy and returns back
to the ground state.
3. Definition
Emission of light by a substance that has absorbed light or other EMR.
Fluorophores: absorption and emission spectrum.
4. • Fluorescence and phosphorescence are the luminescent processes.
• Is associated with the absorption of energy in some form and
emission of the absorbed energy in the form of light.
6. Fluorescence Phosphorescence
Excitation of an electron occurs without the change in the spin. Excitation of an electron occurs with the change in the spin.
Coming back of an electron from the ESS to the GSS is called
as fluorescence
Coming back of an electron from the ETS to the GSS is called as
Phosphorescence
Fluorescence occurs due to singlet singlet transition Phosphorescence occurs due to triplet singlet transition
The life time of an excited state is short ranging from 10-6 to
10-9 seconds
The life time of an excited state is as long as 10-4 seconds to
few seconds or minutes
Fluorescence stops immediately as soon as the source of
excitation is removed
Phosphorescence is seen for a sufficiently longer period, even
after removing the source of excitation.
7. Differentiation
ETS ESS
Less energetic compared to ESS More energetic compared to ETS
Molecule is paramagnetic Molecule is diamagnetic
Transition of an electron to an ETS does involve the
change in spin of electron
Transition of an electron to an ESS does not involve
the change in spin of electron
8. Various deactivation processes
The processes by which excited electrons give out the absorbed energy
& come back to the original position i. e to the ground state in most of
the cases.
1. Vibrational relaxation
2. Internal conversion
3. Fluorescence
4. External conversion
5. Intersystem crossing
6. Phosphorescence
7. Dissociation
8. Predissociation
9. Deactivation Processes
• The loss of energy by an electron, while coming back from an excited
state (S1) to the ground state (S0), is shown by two arrows.
• Wavy arrow: radiation less loss of energy, which is usually in the form
of heat
• Straight arrow: indicates the loss of energy which is usually in the
form of radiation.[Fluorescence & phosphorescence]
10. Deactivation Processes
• As excited molecule can return to the ground state (GS) by a
combination of deactivation processes.
S0: Ground singlet state (GSS)
S1: First excited singlet state (FESS)
S2: Second excited singlet state (SESS)
T1: First excited triplet state (FETS)
Thick lines: Electronic state.
Thin lines: Vibrational energy levels.
11. Vibrational relaxation
• When an electron from the GSS (S0) is excited using a narrow range of
wavelengths, centered around, it occupies any one of the vibrational
energy levels of the FESS.(S1)
• Direct excitation of an electron from the GSS to the first excited triplet
state (FETS,T1) does not occur since it requires a change in the spin of an
electron.
• Deactivation by vibrational relaxation is so fast that the shelf life of an
excited state is only 10
-12 seconds or less.
• It is characterized by the transition of an electron from the higher
vibrational energy level to the lower vibrational energy level, within the
same electronic state. It involves the loss of energy in the form of heat
indicated by wavy arrows.
12. Internal Conversion
• Instead of excitation of an electron from SO, using a wavelength centered around λ1, if
a shorter or a more energetic wavelength centered around λ2 is used, an electron can
get excited to the second excited singlet state (SESS, S2).
• In order to come back to the GS, the electron chooses the fastest deactivation process,
the vibrational relaxation process, by which it comes down from the higher vibrational
energy level of S2 to the ground vibrational energy level V0 of S2.
• An electron can not directly fall down from S2 to any of the vibrational energy levels of
S0 by giving out energy in the form of radiation because it is slower process of
deactivation compared to the other available deactivation processes.
• When one of the vibrational energy levels of the lower excited singlet state (v3 of S1)
overlaps with the ground vibrational energy level (vo) of the higher electronic singlet
state (S2), an electron prefers to deactivate by the process of internal conversion.
• Thus a deactivation process involving transition of an electron from a higher singlet
state to a lower singlet state, through overlapping vibrational energy levels, without
significant loss of energy is termed as internal conversion. The minute loss in the
energy is in the form of heat which is radiation less loss of energy.
13. External conversion
• Like the vibrational relaxation, external conversion is associated with the radiation
less loss of energy i.e. the energy is lost by an excited molecule to its surrounding
(either to the solvent or the neighboring molecules of the solute itself) in the form
of heat.
• External conversion occurs from the lowest excited singlet (S1) and triplet (T1) states
to the ground state.
Vibrational relaxation process External conversion
Transition of an excited electron from the higher vibrational
energy level to the ground vibrational energy level of the
same electronic states
Transition of an excited electron from the ground
vibrational energy level V0 of the higher electronic state to
any of the vibrational energy levels of the lower electronic
state.
Since the transition of an electron is within the electronic
state , the amount of energy given out in the form of heat is
less. Hence there is slight increase in the temperature of
the solvent.
The transition of an electron is from one electronic state to
the other. So the amount of energy given out in the form of
heat is much more compared to that produced during
vibrational relaxation and therefore there is marked
increase in the temperature of the solvent.
14. Fluorescence
• From V0 of S1, it can fall down to any of the vibrational energy levels
of the GS (S0), either by giving out energy in the form of heat
(external conversion) or by giving out energy in the form of radiation
(fluorescence).
• Fluorescence is usually observed at a wavelength which is longer
than the wavelength used for excitation.
• Coming back of an electron from the ESS to the GSS is termed as
fluorescence
15. Intersystem crossing
• Deactivation process is somewhat similar to internal conversion.
• In case of internal conversion, an electron is transferred from the
higher excited singlet state to the lower singlet state (either excited
or ground) without substantial loss of energy and it involves singlet
singlet transition (does not involve change in the spin of an electron)
• Deactivation by intersystem crossing occurs if any of the vibrational
energy levels of a triplet excited state (v2 of T1) overlaps with the
ground vibrational energy level v0 of the lowest excited singlet state
(s1)
• Intersystem crossing involves singlet triplet transition and hence it is
associated with the change in the spin of an electron.
17. Predissociation
• When an electron is excited from S0 to S2 by a shorter wavelength λ2, it
can undergo deactivation, first by vibrational relaxation and then by
internal conversion. This allows entry of an electron from V0 of S2, into
any of the vibrational energy levels of the lower excited electronic
singlet state (S1). This internal conversion sometimes results in
predissociation.
• In a predissociation, an electron enters such a vibrational energy level of
the lower electronic singlet state (S1), whose energy is equivalent to the
energy of one of the bonds in a molecule.
• Hence as soon as an electron occupies that particular vibrational energy
level, it results in the rupture of a particular bond in a molecule. Thus a
part of the absorbed energy is lost in the form of heat (during vibrational
relaxation from the higher vibrational energy level of S2 to V0 of S2.) and
a part of it is used to break a bond in the molecule.
18. Dissociation
• In case of predissociation, the rupture of a bond results as a effect of the
absorption of energy by some chromophore in a molecule, followed by
the internal conversion of the electron into the vibrational energy level
of the lower excited singlet state, which is associated with the bond
energy of one of the bonds in a molecule.
• In case of dissociation, the absorption of energy by an electron directly
takes it to that vibrational energy level whose energy corresponds to
one of the bonds in a molecule and results in the rupture of that
particular bond.
19. Factors affecting intensity of Fluorescence &
Phosphorescence
Two major factors deciding whether a molecule will or will not show
fluorescence or phosphorescence and if yes then what extent
1. Intrinsic structure of molecule
2. Environment of a molecule
The extent to which molecules fluoresce or phosphoresce is
expressed quantitatively in terms of quantum yield or quantum
efficiency. Ø
20. quantum efficiency
Øf = Kf
_______________________________
Kf + Kph +Kic +Kec +Kis +Kpd +Kd
The quantum efficiency for fluorescence is the ratio of the rate of
deactivation of molecules by fluorescence to the rates of all the
deactivation processes followed by it.
21. Intrinsic structure of molecule
• The type of electronic transition involved
i. Saturated compounds involving sigma to sigma star transition
ii. Unsaturated compounds involving pi to pi star or n to pi star
transition
• Aromatic, aliphatic and alicyclic compounds
• The heterocyclic compounds
• The presence of certain substituent's in the Benzene ring
• The rigidity
22. Environment of a molecule
1. Temperature of solution
2. Viscosity of the solution
3. Polarity of the solvent
4. PH of the solution
5. Impurities present in the solution
6. Concentration of the solution
23. Summary (Factors)
Structure:
1.) Aromatic
2.) Rigid structures exhibit more
3.) Heavy atoms will decrease fluorescence
4.) Fluorescence will increase when molecule is adhered to surface
Temperature and Solvent Effects
1.) Lower temperature increases fluorescence
2.) Solvent contains heavy atoms will decrease fluorescence but increase
phosphorescence
25. Instrumentation
• The basic instrument is a spectrofluorometer.
• It contains a light source, two monochromators, a sample
holder and a detector.
• There are two monochromators, one for selection of the
excitation wavelength, another for analysis of the
emitted light.
• The detector is at 90 degrees to the excitation beam.
• Upon excitation of the sample molecules, the fluorescence
is emitted in all directions and is detected by photocell at
right angles to the excitation light beam. 25
26. Instrumentation
• The lamp source used is a xenon arc lamp that emits
radiation in the UV, visible and near-infrared regions.
• The light is directed by an optical system to the excitation
monochromator, which allows either preselection of
wavelength or scanning of certain wavelength range.
26
27. Instrumentation
• The exciting light then passes into the sample chamber
which contains fluorescence cuvette
• A special fluorescent cuvette with four translucent quartz or
glass sides is used.
• When the excited light impinges on the sample cell,
molecules in the solution are excited and some will emit
light.
27
28. Instrumentation
• Light emitted at right angles to the incoming beam is analyzed by
the emission monochromator.
• The wavelength analysis of emitted light is carried out by
measuring the intensity of fluorescence at preselected wavelength.
• The analyzer monochromator directs emitted light of the
preselected wavelength to the detector.
• A photomultiplier tube serves as the detector to measure the
intensity of the light.
• The output current from the photomultiplier is fed to some
measuring device that indicates the extent of fluorescence.
28
29. SOURCE
• Mercury lamp : produces intense radiation but gives a line spectrum.
Under high pressures, lines are obtained at 366 ,405, 436,
446,577,691 and 773 nm
• Xenon lamp: more intense radiation and gives a continuous
spectrum between 250 and 660nm.
30. Filters & Monochromators
• Use of two monochromators
• A primary filter, which selects the wavelength required for the
excitation (λ exe) of a molecule
• A secondary filter, which selects the wavelength of emission (λ em)
• Eg. Interference filters, absorption filters or gratings can be used.
31. Cuvettes / Sample holders
• The cuvettes are usually made up of quartz and contains the solution
of sample to be analyzed.
• Glass or silica cuvettes can also be used but glass absorbs in the
range of 120-350 nm, hence it can not be used if λexe falls in the uv
region.
32. Detectors, Recorder & Printer
• Detectors are usually placed at right angles to the incident beam.
• Photomultiplier tube is mostly used in spectrofluorimetric.
• The intensity of fluorescent can be recorded and printed out using a
printer.
33. Excitation and Emission spectra
• For recording absorption or an excitation spectrum, the substance is excited by
using a number of wavelengths and the emission of radiation is measured at a
fixed wavelength.
• The wavelength at which a substance absorbs the maximum is referred to as
excitation wavelength (λ exe) and it should be used
for the measurement in order to excite the maximum number of molecules and
to get the maximum intensity of emission.
• The emission spectra are recorded by exciting the molecules at a fixed
wavelength and recording the intensity of emission at varying wavelengths.
• The wavelength at which the molecules give out maximum energy is called as
emission wavelength.[λ em]
34. QUENCHING OF FLUORESCENCE
• The reduction in or the total loss of intensity of fluorescence is called
quenching.
• Environmental factors responsible for quenching of fluorescence are
i. Temperature of the solution
ii. Viscosity of the solution
iii. Polarity & PH of the solution
iv. Presence of any paramagnetic species in the solution
v. Concentration of the solution
vi. The self absorption
35. Temperature of the solution
• Increase in temperature of the solution increases the number of
collisions among the neighboring molecules (solute-solute or solute-
solvent).
• This reduces the intensity of fluorescence and brings about
quenching of fluorescence.
36. Viscosity of the solution
• Decrease in viscosity of the solution also increases the number of
collisions among the neighboring molecules, thereby causing
quenching of fluorescence.
37. Polarity and pH of the solution
• Decides the wavelength of maximum absorption of a solute.
• Any solvent or the change in PH, which brings about the change in
the wavelength of maximum absorption of a solute results in the
decrease in the intensity of fluorescence.
38. Presence of any paramagnetic species in the
solution
• Like oxygen or the presence of halides or compounds containing
heavy atoms in solution also decreases the intensity of fluorescence.
39. Concentration of the solution
• The use of highly concentrated solution reduces intermolecular
distance. This increases the collisions among the molecules.
• The loss or decrease of fluorescence due to collisions between the
excited molecules of solute is called as self quenching.
• The increase in concentration of a solute increases the extent of self
quenching.
40. The self absorption
• The self absorption occurs when the wavelength of emission overlaps
the wavelength of absorption i.e. whatever energy is absorbed by the
compound, the same is emitted out by it and is absorbed by the
other molecules of the same sample in the solution.
41. Application of Spectrofluorimetry
• Is used for the analysis only if the compounds are fluorescent in
nature.
• Compounds containing following features usually show a property of
fluorescence
i. A conjugated pie system
ii. Some extent of rigidity
iii. Absence of electron withdrawing groups such as –COOH or NO2
42. Application of Spectrofluorimetry
UV-Visible Spectrometry Spectrofluorimetry
Is chosen when the
concentration of an
analyte is not very low
and there is no
interference from many
substances.
Is chosen when an
analyte is in low
concentration and there
are many interfering
substances in the sample
to be analyzed.
The quantitative analysis in spectrofluorimetric is
carried out by setting the excitation wavelength at
the maximum absorption and setting the emission
wavelength at the maximum intensity of emission.
43. Quantitative analysis by Spectrofluorimetry
• Direct methods: which are used for the compounds that are naturally
fluorescent
• Derivatization methods: which are used for the compounds that are
non fluorescent in nature
• Quenching methods: which are used for the compounds that are
able to quench the intensity of fluorescence of some fluorescent
species.
• Fluorescence and chromatography
• Fluor immunoassays
44. Measurement of Fluorescent Intensity: Fluorimeters or Spectrofluorometers
Spectrometers Spectrofluorometers
Uses only one monochromator Use of two monochromators
First monochromator is used to isolate the
excitation wavelength i.e. wavelength
required for the excitation of fluorescent
molecule.
Second monochromator is required to
isolate the emission wavelength i.e.
wavelength emitted by the molecule while
coming back from an excited state to the
ground state.
Position of detectors: the intensity of the
emitted beam is measured along the
direction of the path of the incident beam
The intensity of the emitted beam is
measured at an angle preferably at 90
degree, with respect to the direction of the
path of the incident beam
The radiation of fluorescence is emitted in all the directions, but the intensity is
usually measured at 90 degree to the incident beam ,the intensity of fluorescence
is maximum and so accuracy of the measurement is also maximum. In other
directions the intensity is reduced due to the increased scattering from the
solution and also from the walls of the cuvette or sample holder.