Fluorometry PPT file.ppt

Principle of fluorescence and phosphorescence

1. Fluorescence spectroscopy
(Fluorometry)
When certain chemical substances are excited
electronically by absorption of UV or visible radiation,
they emit light at a longer wavelength. This phenomenon
is called luminescence.
Depending upon the life span of the excited species
luminescence can be divided in to two types-
i)Fluorescence and
ii)Phosphorescence

2. Fluorescence: If the luminescence stops within 10
-8
to
10
-4
seconds, it is called fluorescence.
Phosphorescence: If the luminescence continues for a
longer period of time (10
-4
to 10 seconds), it is called
phosphorescence.

3. Theory:
When a sample is irradiated by ultraviolet or
visible light, molecules are excited
electronically. Each excited electronic states
has many different vibrational energy levels
and excited molecules will be distributed in
the various vibrational energy levels of the
excited states.

4. •Most usually this state is a singlet state.
•In the singlet state, all the electrons are
paired, and in each pair, the two electrons
spin about their own axes in opposite
directions.
•Molecules from the excited singlet state can
lose energy by several mechanisms and
return to the ground state.

5. If the excited molecules emit radiation at the same
wavelength the process is termed resonance fluorescence
which is very rare.
More usually molecules undergo a radiationless loss of
vibrational energy and quickly fall to the lowest vibrational
level of the excited state. The vibrational energy is thought
to be lost to solvent molecules.
From the lowest vibrational energy level of the excited state, a
molecule can return to the ground state by photo emission
and the process is known as fluorescence.
.

6. •Because of vibrational relaxation, the radiation
emitted as fluorescence is of lower energy and
therefore of longer wavelength than that originally
absorbed.
•An excited molecule may loss energy by other
processes. It may for example, undergo a
radiationless loss of energy sufficient to drop to the
ground state. This process is termed internal
conversion.

7. With some compounds a process known as intersystem
crossing can also occur. Here a molecule in the lowest
vibrational level of the excited state converts to a triplet
state.
The triplet state lies at an energy level intermediate
between ground and excited states and is
characterized by an unpairing of two electrons.
Thus, in contrast to the singlet state, there is a spin
reversal of one electron and the two electrons spin
about their own axes in the same direction.

8. Once intersystem crossing has occurred, a molecule
quickly drops to the lowest vibrational level of the
triplet state by vibrational relaxation.
The triplet state is much longer lived than the
corresponding singlet state with lifetimes of 10
-4
to
10 seconds.
From the triplet state a molecule can drop to the ground
state by emission of radiation. This type of luminescence
is termed phosphorescence.

9. •Phosphorescence is often characterized by an
afterglow i.e., because of the long life of the triplet
state luminescence can be observed after the source
of exciting radiation has been removed.
•In contrast, no afterglow is observed in fluorescing
systems because of the short life of the excited state.
•These processes are diagrammatically shown in fig. 1.

10. •Thus fluorescence may be defined as the radiation
emitted in the transition of a molecule from the
excited singlet state to the ground state while
phosphorescence is the radiation emitted in the
transition of a molecule from the excited triplet state
to the ground state.

11. Fig.1The energy level diagram showing various electronic processes

12. Molecular structure and fluorescence
Chromophorelightabsorptionfluorescence
Again,definitecorrelationbetweenchemicalstructureand
fluorescencecannotbemadedueto
radiationlessprocessesand
intersystemcrossing.
However,itisusuallyexpectedthatthestructuralfeatureswhich
influencethedegreeofconjugationofamoleculeand
thedelocalizationofpelectrons,
mightinfluencetheintensityoffluorescence.

13. Thus
saturatedcompoundsarenonfluorescent.
Examplesarebutane,pentane,hexane,cyclohexaneetc.
Unsaturatedcompounds likebenzeneisweakly
fluorescentand
Polycyclicaromaticcompoundsarestronglyfluorescent.
Examplesareanthracenes.
Similarly,riboflavinisfluorescent
reducedriboflavinisnonfluorescent
wheredegreeofconjugationisreduced.

14. Riboflavin
Reduced riboflavin

15. Molecular geometry also influences the intensity of
fluorescence.
For example, trans-stilbene is a planar molecule and is
more fluorescent than the nonplanar cis-stilbene.
trans-stilbene cis-stilbene
Planar non-planar
more fluorescent less fluorescent

16. Instrumentation:
Fluorometer/spectrofluorometer
Thecomponentsofafluorometerorspectrofluorometer
arequitesimilarindesignandfunctiontothoseemployed
incolorimeterorspectrophotometer.
Thechiefcomponentsofafluorometerare
i)a radiation source
ii)an excitation filter or monochromator
iii)a sample holder
iv)an emission filter or monochromator
v)a detector and
vi)a recorder

17. Radiation source
Must be very intense and stable
Commonly used lamps are:
Mercury discharge and xenon lamp.
Excitation filter
The function of excitation filter is to isolate a band of
exciting light.
A glass filter is usually used.

18. Sample holder
Glass cells are adequate for most fluorescence analysis.
Quartz cells are used below 320nm.
Emission filter
Select a band of fluorescence for detection.
It is placed at right angle to the beam of excitation light.
Detector
Photomultiplier tube is used as a detector.

19. Recorder
Theoutputofthedetectorisconnectedtoameter,adigital
displayorarecorder.

20. Factorsinfluencingtheintensityoffluorescence
1.Concentrationoffluorescingspecies
2.Solvents
3.Presenceofothersolutes
4.Hydrogenionconcentration/pHofthesolution
5.Temperatureetc.

21. 1.Concentrationoffluorescingspecies
butthisrelationshipismorecomplexthantherelationshipbetween
Insomecases,photonsemittedasfluorescencecanbeabsorbedin
excitingothermoleculesandwillnotbemeasuredasfluorescence.
2.Solvents
Thespectralcharacteristicsoffluorescedlightcanvaryfrom
solventtosolventdueto
polarizationeffectsandhydrogenbonding.

22. 3.Presenceofothersolutes
caninfluencetheintensityoffluorescencebyanumberofways,
suchas
a)impuritieseffect
b)innerfiltereffect
c)chemicalquenching
a)Impuritieseffect:
Impuritiesinasamplefromsolvents,buffersorglasswarescan
influencetheintensityoffluorescence.Theimpuritiescanfluoresce
andthuscanintroduceanerrorindeterminationoffluorescence.

23. b)Innerfiltereffect:
Thesolutemayabsorbeithertheexcitationoremissionradiationand
thusreducestheintensityoffluorescence.
c)Chemicalquenching:
Chemicalquenchingistwotypes
i.collisionalquenchingand
ii.staticquenching

24. 4. Hydrogen ion concentration / pH of the solution
Weak acid / weak bases
Phenol / α-naphrhol
5.Temperature
At higher temperature radiationlessprocesses are favored. Since

25. Comparison of fluorometrywith spectrophotometry
Fluorometry Spectrophotometry
1. Fluorometryis more sensitive as
an analytical tool than is
spectrophotometry
1. Spectrophotometryis less
sensitive.
2. It is more specific in identifying
and analyzing a compound than
spectrophotometry.
2. It is less specific.
3. Temperature must be maintained
reasonably controlledin a
fluorometricmethod.
3.Variation of temperature does
not affect much in a
spectrophotometricanalysis.
4.Intensity of incident light must be
stable
4.Intensity of incident light need
not be rigidly controlled in a
spectrophotometricprocedure.

26. Fluorometry Spectrophotometry
5. Impurities or other extraneous
solutes can markedly affect the
intensity of fluorescence by
quenching effect.
5. The absorbance of a compound
is not significantlyaltered by the
presence of other solutes.
6. The intensity of fluorescence of
many drugs is pH dependant.
6. Spectrophotometricanalysis is
not affected much by small
variation of pH of the solution.

27. Applicationoffluorometry
1.Widerangeofapplication
•apowerfultoolforstudyingmolecularinteractionsin
•analyticalchemistry,biochemistry,physiology,cell
biology,cardiologyandenvironmentalscience.
Manypharmaceuticals/drugsarefluorescentcompoundsand
canbeassayedfluorometrically.Forexampleriboflavin,
quinine,salicylatesetc.
Somenonfluorescentdrugscanalsobeassayed
fluorometricallybyconvertingthemintofluoresentderivatives.

28. 2.Sensitivity
Concentrationsaslowas10
-7
Mcanbemeasuredaccurately.
3.Selectivity/specificity
Fisveryspecific&thereforeusefulintheanalysisoftrace
amountofdrugsandmetabolites
inblood,urineandotherbiologicalfluids
andthushelpsinthestudyof
ratesandmechanismof
drugabsorption,metabolismandexcretion.
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