Fluorometry Manik

Introduction, theoretical principle, quantum efficiency of fluorescence, molecular structure of
fluorescence, instrumentation, factors influencing the intensity of fluorescence, comparison of
fluorometry with spectrophotometry, application of fluorometry in pharmaceutical analysis

2. Luminescence:Luminescenceisthephenomenonofa
chemicalspeciestoabsorbradiationofUVorvisible
regionandemitaradiationoflongerwavelength.Lossof
energyandconcomitanttransitionofmoleculesfrom
excitedstatestogroundstateswithemissionofradiationis
calledluminescence.
Luminescencecanbedividedintotwotypesdepending
onthelifespanoftheexcitedstate–
1.Fluorescence
2.Phosphorescence

3. Singlet and triplet states
•Ground state –two electrons per orbital; electrons have opposite spin and are paired
Singlet excited state
Electron in higher energy orbital has the opposite spin orientation relative to
electron in the 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.
Typesofemission
Fluorescence –return from excited singlet state to ground state; does not require change in
spin orientation (more common of relaxation)
An atom or molecule that fluoresces is termed a Fluorophore
Fluorometry is defined as the measurement of the emitted fluorescence light
Phosphoresence –return from a triplet excited state to a ground state; electron requires change
in spin orientation

4. Vibrationalenergylevel:Whetherthemoleculeisingroundstateor
excitedstate,themoleculecontainsmanyenergylevelswhicharecalled
vibrationalenergylevels.
Vibrationalrelaxation:Vibrationalrelaxationisthetransitionofmolecule
fromanyofthevibrationalenergylevelstothelowestvibrationalenergy
leveloftheexcitatorystate.
Resonancefluorescence:Resonancefluorescenceisthephenomenon
wherethemoleculeabsorbsandemitsequalamountofenergy.Practically
resonancefluorescencedoesn’toccuroroccurrarelyasvibrational
relaxationoccurs.
Internalconversion:Thephenomenonofexcitedmoleculetoreturntothe
groundstatebylosingenergybymeansotherthanphotoradiationis
termedinternalconversion.
Intersystemcrossing:Thetransferofamoleculepresentinthelowest
vibrationalenergyleveloftheexcitedsingletstatetoanexcitedtripletstate
iscalledintersystemcrossing.

5. Property Fluorescence Phosphorescence
Transition Moleculetransitsfromexcitedsingletstate
togroundstate.
Moleculetransitsfromexcitedtriplet
statetogroundstate.
Lifespan Fluorescenceiscontinuedforonly10
-8
to10
-
4
seconds.
Phosphorescencecontinuesfor10
-4
secondsto10seconds.
Afterglow Notpresent. Occursandluminescenceslowlyfades.
Analytical applicationYes. No.
Quantumefficiency:Quantumefficiencyisdefinedastheratioofnumberoflightquantaemittedandthe
numberoflightquantaabsorbed.
Itssignificanceisthat,itisanindicatorofhowfluorescentamoleculeis.IfQisnear1,themoleculeishighly
fluorescentmoleculeandifQisnear0,themoleculeisaverylowfluorescentmolecule.absorbedlight ofEnergy
emittedlight ofEnergy
absorbed quantalight of No.
emitted quantalight of No.
orQ

6. Themethodofanalysingasamplebymeasuringitsfluorescencei.e.
intensityandcompositionoflightemittedbyit,iscalledfluorometry.
Fluorescencespectroscopyakafluorometryorspectrofluorometryisan
analyticaltechniqueforidentifyingandcharacterizingminuteamountsofa
fluorescentsubstancebyexcitationofthesubstancewithabeamof
ultravioletlightanddetection&measurementofthecharacteristic
wavelengthofthefluorescentlightemitted.
Itisaspectrochemicalmethod.

7. WhenenergyisappliedtocertainmoleculesintheformofUVorvisible
electromagneticradiation,themoleculestemporallytransittoan
excitedsingletstatewheretheexcitedelectronisinpairedcondition
withthegroundelectron.Intheexcitedstate,themoleculesloseenergy
inradiationlessmannertodescendtothelowestvibrationalenergy
leveloftheexcitedstate.Theexcitedstatelastsonly10
-8
to10
-4
seconds
andthentheexcitedmoleculewillreturntogroundstatebylosing
energythroughemittingradiation.Thisistermedfluorescenceandthe
emittedradiationisoflongerwavelength.
Bymeasuringtheemittedwavelengthwecandeterminethepresence
andamountofacompoundinasample.

9. Relationship between fluorescence and
chemical structure
Definite correlations between chemical structure and fluorescence can’t be made.
Degreeofconjugation
Delocalizationofelectron
Electrondonatinggroups
Exception

10. Relationship between fluorescence and
chemical structure
•Electron withdrawing
groups
•Exception

11. Relationship between fluorescence and
chemical structureMolecular geometry
Rigidity and planarity
cis-trans isomerism
Heterocyliccompounds
Ionization
Complexation

13. ISSPC1(ISSInc.,Champaign,IL,USA)
Fluorolog-3 (Jobin Yvon Inc, Edison, NJ, USA )
QuantaMaster (OBB Sales, London, Ontario N6E 2S8)

14. Concentration of fluorescing species
Presence of other solutes/impurities
Chemical quenching
pH of the sample solution
Stability of the sample compound (Degradation of Sample)
Solvent effect
Temperature

15. Features Absorption spectroscopy Fluoroscence spectroscopy
Theoretical consideration
Measurement ofamountoflight
absorbed.
Measurementofintensityoffluorescence.
Wavelength of light usedWhichgivesmaximumabsorption. Whichgivesmaximumfluorescence.
Instruments Determinesonlytheabsorptionoflight.
Determinesabsorptionoflightaswellas
emissionofradiation.
Light source Tungsten,H
2-dischargelamp. Mercuryarclamp,Xenonarclamp.
Cell used Silicacell. Glassandmetalcells.
Detector
Phototubeorphotomultiplierisusedto
detecttheradiationabsorbed
Emissionfilterisusedtoseparatethe
emittedlightfromthetransmittedlight.
Concentration
Concentrationdependsonthemolar
absorptivity.
Concentration depends on the
characteristicsoftheinstrument.
Electrical transition
Applicableforbothππ*&nπ*
transition.
Notapplicableforthecompound
containingnπ*transition.
Experimental variables
temperature & Extraneous
solution
Notsorestricted. Highlyrestricted.
Sensitivity and selectivityLesssensitiveandlessspecific. Moresensitiveandhighlyspecific.

16. Application in chemistry
Determination of metal ions
Separation and identification
Application in biopharmaceutics
Pharmaceutical applications

17. Sensitivity
Specificity
Wide Concentration Range
Simplicity and Speed
Low Cost
Limitations of Fluorometry

18. Excitation spectrum and emission spectrum
Theexcitationspectrumisameasureoftheabilityoftheimpingingradiationtoraisea
moleculetovariousexcitedstatesatdifferentwavelengths.Anexcitationspectrumisrecording
offluorescenceversusthewavelengthoftheexcitingorincidentradiationanditisobtainedby
settingtheemissionmonochromatortoawavelengthwherefluorescenceoccursandscanningthe
excitationmonochromator.Anexcitationspectrumlooksverymuchlikeanabsorptionspectrum,
becausethegreatertheabsorbanceattheexcitationwavelength,themoremoleculesarepromotedto
theexcitedstateandthemoreemissionwillbeobserved.
Theemission(fluorescence)spectrumisameasureoftherelativeintensityofradiationgiven
offatvariouswavelengthasthemoleculereturnsfromtheexcitedstatestothegroundstate.
Theemissionspectrumisrecordingoffluorescenceversusthewavelengthofthefluorescence
radiation,anditisobtainedbysettingtheexcitationmonochromatortoawavelengththatthesample
absorbsandscanningtheemissionmonochromator.
Sincesomeoftheabsorbedenergyisusuallylostasheat,theemissionspectrumoccursatlonger
wavelengths(lowerenergy)thandoesthecorrespondingexcitationspectrum.Ifanemissionspectrum
occursatshorterwavelengthsthantheexcitationspectrum,thepresenceofasecondfluorescing
speciesisconfirmed.
Theabsorptionandemissionspectrawillhaveanapproximatemirrorimagerelationshipifthe
spacingsbetweenvibrationallevelsareroughlyequalandifthetransitionprobabilitiesaresimilar.

19. Energyleveldiagramshowingwhystructureisseenintheabsorptionandemissionspectra,andwhythespectraseem
roughlymirrorimagesofeachother.

20. Mirror image rule
•Vibrational levels in the excited states and ground states are
similar
•An absorption spectrum reflects the vibrational levels of the
electronically excited state
•An emission spectrum reflects the vibrational levels of the
electronic ground state
•Fluorescence emission spectrum is mirror image of
absorption spectrum
S
0
S
1
v=0
v=1
v=2
v=3
v=4
v=5
v’=0
v’=1
v’=2
v’=3
v’=4
v’=5

21. References
•Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York.
•Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York. 3 Id., p. 7.
•Dr.Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular Biology, School of Medicine.
•G. K. Turner, "Measurement of Light From Chemical or Biochemical Reactions," in Bioluminescence and Chemiluminescence: Instruments and
Applications, Vol. I, K. Van Dyke, Ed. (CRC Press, Boca Raton, FL, 1985), pp. 45-47.
•Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 51-57.
•Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, chap. 2.
•Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 67-69.
•Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, pp. 23-26.
•Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 57-58.
•Stotlar, S. C. 1997. The Photonics Design and Applications Handbook, 43rd Edition, LaurinPublishing Co., Inc., Pittsfield, MA, p. 119.
•Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 63.
•Dr.Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular Biology, School of Medicine.
•Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 30.
•Dr.Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular Biology, School of Medicine.
•Iain Johnson, Product Manager, and Ian Clements, Technical Assistant Specialist (May 1998 communication from Molecular Probes, Eugene,
Oregon).
•FluorometricFacts: A Practical Guide to Flow Measurement, Turner Designs (1990), pp. 14-15.
•Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 172.
•FluorometricFacts: A Practical Guide to Flow Measurement, Turner Designs (1990), p. 21.
•Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York., p. 28.
•TeitzTextbook of Clinical Chemistry and Molecular diagnosis (5th Edition)
•Dr.B.K.Sharma, Instrumental methods of chemical analysis.
•GurdeepR Chatwal, Instrumental methods of chemical analysis
•http://en.wikipedia.org/wiki/Fluorescence
•http://images.google.co.in/imghp?oe=UTF-8&hl=en&tab=wi&q=fluorescence
•http://www.bertholdtech.com/ww/en pub/bioanalytik/biomethods/fluor.cfm