This document provides an overview of fluorescence spectroscopy. It defines luminescence as the emission of photons from electronically excited states. There are two main types of luminescence: fluorescence from singlet excited states and phosphorescence from triplet excited states. The document discusses instrumentation for fluorescence spectroscopy including light sources, wavelength selection devices like filters and monochromators, and detectors. It also covers factors that affect fluorescence intensity such as molecular structure, concentration, temperature, and pH.

1. 1
By: Bijaya Kumar Uprety
Fluorimetry

2. LUMINESCENCE SPECTROSCOPY
•The emissionof radiation from a species after that species has absorbed radiation.
LUMINESCENCEFLUORESCENCEPHOSPHORESCENCESPECTROSCOPYCHEMILUMINESCENCE
2

3. What is luminescence ?
Luminescenceistheemissionofphotonsfromelectronicallyexcitedstate.
Luminescenceisdividedintotwotypes,dependinguponthenatureofthegroundandtheexcitedstates.
Inasingletexcitedstate,theelectroninthehigherenergyorbitalhastheoppositespinorientationasthesecondelectroninthelowerorbital.Thesetwoelectronsaresaidtobepaired.Returntothegroundstatefromanexcitedsingletstatedoesnotrequireanelectrontochangeitsspinorientation.
Inatripletstatetheseelectronsareunpaired,thatis,theirspinshavethesameorientation.Achangeinspinorientationisneededforatripletstatetoreturntothesingletgroundstate.
diamagnetic S1
paramagnetic T1
So

4. LUMINESCENCE SPECTROSCOPY
Absorption first - Followed by emission in all directions , usually at a lower frequency 4

5. Types of luminescence
(classification according to the means by which energy is supplied to excite the luminescent molecule)
1)Photoluminescence: Molecules are excited by interaction with photons of radiation.
Fluorescence:
Prompt fluorescence: S1S0 + h
The release of electromagnetic energy is immediate or from the singlet state.
Delayed fluorescence: S1T1S1S0 + h
This results from two intersystem crossings, first from the singlet to the triplet,
then from the triplet to the singlet.
Phospholuminescence:T1S0+ h
A delayed release of electromagnetic energy from the triplet state.
2) Chemiluminescence: The excitation energy is obtained from the chemical energy of
reaction.
3) Bioluminescence: Chemiluminescence from a biological system: firefly, sea pansy, jellyfish, bacteria, protozoa, crustacea.
4)Triboluminescence: A release of energy when certain crystals, such as sugar, are broken.
5)Cathodoluminescence: A release of energy produced by exposure to cathode rays
6)Thermoluminescence: When a material existing in high vibrational energy levels emits energy at a temperature below red heat, after being exposed to small amounts of thermal energy.

6. LUMINESCENCE SPECTROSCOPY
•Collectively,fluorescenceandphosphorescenceareknownasphotoluminescence.
•Athirdtypeofluminescence-Chemiluminescence-isbaseduponemissionoflightfromanexcitedspeciesformedasaresultofachemicalreaction.
•Infavorablecases,luminescencemethodsareamongstsomeofthemostsensitiveandselectiveofanalyticalmethodsavailable.
•DetectionLimitsareasageneralruleatppmlevelsforabsorptionspectrophotometryandppblevelsforluminescencemethods.
•Mostchemicalspeciesarenotnaturallyluminescent.
•Derivatisationreactionsareoftenavailabletoformluminescentderivativesofnon-luminescentcompounds.
•However,thisextrasteplessenstheattractivenessofluminescencemethods.
•Fluorimetryisthemostcommonlyusedluminescencemethod.
•Thetermsfluorimetryandfluorometryareusedinterchangeablyinthechemicalliterature.
6

7. Introduction
•Alargenumberofsubstanceabsorbultravioletandvisiblelightenergy.But,therearesomesubstanceswhichloseexcessenergyasheatthroughcollisionswithneighboringatomsormolecules.
•Theenergyemittedbythesesubstanceshasawavelengthlargerthanthatofabsorbed.Thisprocessofemittingradiationwithlargerwavelengththanthatofabsorbedisknownasluminiscence.
•Luminiscenceismainlyoftwotypes:
1.Fluorescenceand2.Phosphorescence.
7

8. Concept of singlet and triplet state
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

9. 9

10. Fluorimetry
•Fluorimetryisananalyticalmethodforthemeasurementoffluorescence,baseduponemissionofabsorbedradiationbythemolecules.Whenamoleculeabsorbsincidentelectromagneticradiations,itisexcitedtohigherenergylevel,whereitisunstable.
•Therefore,itreturnstothegroundstatebyemittingtheabsorbedradiations.Whentheemissionoccursdirectlyfromsingletexcitedstatetosingletgroundstatewithoutanytransitionsandwithoutanychangeinspinorientation,itisreferredasfluorescence.
•Andwhentheemissionoccurswithinafractionofsecondtofewdaysofabsorptionofradiationsduetotransitionfromsingletexcitedstatetotripletstateandthentosingletgroundstate,itisreferredasphosphorescence.Itinvolvesthechangeinspinorientationduringthetransitionfromexcitedsinglettotripletandfromtriplettosingletgroundstate.
•Themovementofelectronsfromexcitedsingletstatetotripletstate,iefromunpairedelectronswithoppositespintounpairedelectronswithsamespinistermedasintersystemcrossing.
•Fluorescenceandphosphorescencearecombinedlycalledphotoluminescence.
•Theenergyemittedbythesemechanismhasawavelengthlargerthanthatofabsorbed.
10

11. •Thebasisoffluorimetryisthemeasurementoffluorescence.Drugswhichareintrinsicallyfluorescent,aredeterminedfluorimetrically.
e.g.Quininesulfatein0.1Nsulfuricacid;Ergometrinein1%tartaricacidetc.
11

12. Principle of fluorimetry
•Amoleculeabsorbsincidentelectromagneticradiationsandgetexcited.
•Itisunstableinitsexcitedstateandtendstoreturntothegroundstatebyemittingradiation.
•Fluorescencecanbereferredastheradiationsemittedfromanexcitedmoleculeintransitionfromsingletexcitedstatetosingletgroundstate.
•Influorimetry,radiationsemittedareoflongerwavelengththanabsorbedradiations.Thisisbecause,whentheradiationsfallonthemolecule,vibrationoccurs(in10-13seconds),andhasanaveragelifeof10-9seconds.Duringthevibrationperiod,lossofenergyoccursduetointermolecularcollisionsandsomeenergyislosttosolventmolecules(moleculesofthesolventusedforthedissolutionofthesampleinfluorimetry).Hencetheemittedradiationsareoflongerwavelengthandhavelessenergy.(sinceEαc/λ)
12

13. •Orbital changes can be explained as:
1.Fluorescence:
Singlet state Singlet excited state singlet ground state
pp* (No change in spin)
2. Phosphorescence:
Singlet state Singlet excited state Triplet state (2 unpaired electrons) singlet ground state
pp* (No change in spin) Change in spin
•In either fluorescence or phosphorescence the frequency of the emitted radiation is less than the frequency of incident radiation. The relationship is ;
0> fluor> phos
Where,  = frequency 13

14. Fluorescence related to concentration
ThefluorescenceradiantpowerFisproportionaltotheabsorbedradiantpower.
F=(Po–P)
where=fluorescenceefficiency,Po=incidentpower,P=transmittedpower
TherelationshipbetweentheabsorbedradiantpowerandconcentrationcanbeobtainedfromBeer’slaw.
P/Po=10–A=10–bCor,P=Po10–bC
or,F=Po(1–10–bC)------------(1)
This is the fluorescence law.
WhenexpandingtheexponentialtermsandassumingbCtobe0.05orless, onlythefirsttermintheseriesissignificantandequationcanbewrittenas
F=Po(lnbC)=kbC-----------(2)

15. •wherekisaconstantequaltoPoln.Thus,whentheconcentrationsareverydiluteandnotover2%oftheincidentradiationisabsorbed,thereislinearrelationshipbetweenfluorescentpowerandconcentration.
•WhenbCisgreaterthanabout1.5,10–bCismuchlessthan1andfluorescencedependsdirectlyontheincidentradiationpower.
F=Po
•Fromequation2,wecouldinferthatfluorescenceintensityisdirectlyproportionaltoconcentration.Anotherimportantresultcanbeimpliedfromequation2wherefluorescenceisshowntobedirectlyproportionaltotheintensityoftheincidentbeam.Thissuggeststhataveryintenselightsourceisnecessaryforfluorescenceinstrumentation.Alsosubstancesoflargearepotentialfluorescentmoleculesandshouldbesoughforbetterresults.
15

16. Po
Concentration of fluorescing species
Theoretical behavior of fluorescence as a function of concentration.

17. Factors affecting fluorescence
1.Natureofmolecules:
•Themoleculeswhichhavethetendencytoabsorbelectromagneticradiations(ieUV-Vis)canonlyexhibitfluorescence.
•Unsaturatedmoleculeswithpibondsandgoodresonancestabilitycanexhibitfluorescence.E.g.alkeneswithconjugateddoublebond.
•Saturatedmoleculeswithsigmabondsdonotexhibitfluorescence.E.g. aliphaticandsaturatedcyclicorganiccompounds.
•Ingeneral,thegreatertheabsorbancyofamolecule,themoreintenseitsluminiscence.
2.Affectoffunctionalgroups:Functionalgroupsexhibitmarkedeffectonfluorescenceoftenly.
•Electrondonatinggroupsimprovefluorescence.E.g.amine,hydroxy,andmethoxygroups.
•Electronacceptinggroupsdeteriorateorcompletelydestroyfluorescence.E.g.carboxylic,nitroandazogroup,halidesetc
17

18. 3.Highmolecularweightcompounds:
Ifanelectronofanatomwithhighmolecularweightisexcited,itexhibitsdecreasedfluorescence.
4.Effectofconcentration:
•Thereexistsalinearrelationshipbetweentheconcentrationofsampleandfluorescenceuptoanabsorbancevalueof0.02.Thelinearityholdsgoodtoabout5%ofcaseswithabsorbancesupto0.05.
•Indilutesolution,theradiationdistributeuniformlythroughoutthesolutionandgetabsorbeduniformlygivinghighintensefluorescence.
•Inhighlyconcentratedsolution,upperlayersofthesolutionabsorbmoreradiationsandlessamountofradiatonsaretranserredtolowerlayers.Thus, thereisnouniformradiationsabsorptionwhichresultsindecreasefluorescence.
•Inhighlyconcentratedsolution,intramolecularcollisionscauselossofvibrationalenergyandcertainamountoffluorescecewhichisemittedisreabsorbedresultingindecreaseinintensityoffluorescence.Thisiscalledasconcentrationquenching.
5.Effectofoxygen:Thereisdecreaseinfluorescenceinthepresenceofoxygendueto
•Directconversionoffluorogenicmaterialintonon-fluorogenicmaterial.
•Indirectlyduetoquenching.‘Quenchingreferstothedecreaseinintensityoffluorescenceasaresultofdecreaseinthesensitivityofconstituents.
18

19. 6.AffectofpHofthesolution
•Dependingupontheacidityoralkalinity,asubstancecanbeionizedorunionizedandhence,canbefluorogenicornon-fluorogenic.
e.g.Phenolinneutralandalkalinemediumundergoesionisationandgivesweakfluorescence,whereasinacidmediumitisunionisedandgivesintensefluorescence.
7.AffectofTemperatureandViscosity
•Alterationintemperaturemayaffecttheconcentrationandviscosityofthesample.
•Increaseintemperaturemayresultinincreaseinconcentrationanddecreaseinviscosityresultinginintermolecularcollisionsanddeactivation ofexcitedmoleculedestroyingfluorescence.
•Somesubstancesmayexhibitfluorescenceattemperaturelowerthanroomtemperatureorinaviscoussolventorglassymatrix.
8.Affectofimpurities:Substancesotherthanthesolutemoleculesareimpuritiesandexhibitfluorescencequenching.E.g.iodideionisextremelyeffectivequencher.
19

20. 9.Affectofintensityofradiation:
•Radiationofadequateintensitymustbeusedtoinducefluorescence.
•Highintensityradiationcausesdecreaseinfluorescenceduetophotochemicalchange.
•Lightofsinglewavelength,i.e.monochromaticlightshouldbeused,asenergyofradiationvarieswithwavelength.
10.ChemicalQuenching:
Thiscanoccurintwoways;
•Theexcitedmoleculetransfersitsfluorescentintensitytosurroundingmolecules,ionsorimpuritybyintercollisions,therebydestroyingfluorescence.
•Theunexcitedmoleculemayformastablecomplexwithquenchermoleculeinhibitingexcitationandfluorescence.
11.Affectofstructureofthecompounds:
•Closedringaromaticcompoundsexhibitfluorescencee.g.fluorescein, Eosin.
20

21. •Compoundswithmorethantwocyclicstructureexhibitsfluorescence.E.g. vitaminK,Nucleosides,purines.
•Rigidmoleculeslikemetalcomplexeshaveenhancedfluorescenceastheyinhibittheliberationofexcitationenergy.
•Thepositionoffunctionalgroup(chromophore)whichisresponsibleforabsorptionaffectsfluorescence.
21

22. Instrumentation
•Thebasicdesignofinstrumentationformonitoringmolecularfluorescenceandmolecularphosphorescenceissimilartothatfoundforotherspectroscopies.Themostsignificantdifferencesarediscussedinthefollowingsections.
•MolecularFluorescenceAtypicalinstrumentalblockdiagramformolecularfluorescenceisshowninFigure10.45.Incontrasttoinstrumentsforabsorptionspectroscopy,theopticalpathsforthesourceanddetectorareusuallypositionedatanangleof90°.
•Twobasicinstrumentaldesignsareusedformeasuringmolecularfluorescence.Inafluorometertheexcitationandemissionwavelengthsareselectedwithabsorptionorinterferencefilters.Theexcitationsourceforafluorometerisusuallyalowpressuremercuryvaporlampthatprovidesintenseemissionlinesdistributedthroughouttheultravioletandvisibleregion(254,312,365,405,436,546,577,691,and773nm).
•Whenamonochromatorisusedtoselecttheexcitationandemissionwavelengths, theinstrumentiscalledaspectrofluorometer.Withamonochromator,theexcitationsourceisusuallyahigh-pressureXearclamp,whichhasacontinuumemissionspectrum.
22

23. Instrumentation for fluorescence spectroscopy
Power
supply
Source
Excitation monochromator
Emission monochromator
Detector
Sample cell
Slit
Data processor
General layout of fluorescence spectrophotometer

24. Schematic diagram of a typical spectrofluorometer.

25. 1) Light sources
a. Gas discharge lamps :
Xenon arc lamp
High pressure mercury vapor lamp
b. Incandescent lamps : Tungsten wire filament lamp
c. Laser : tunable dye laser
d. X-ray source for X-ray fluorescence
2) Wavelength selection devices
a. Filters :
Absorption filters ---tinted glass or gelatin containing dyes sandwiched between glass
Interference filters ---thin transparent layer of CF2or MgF2sandwiched two parallel,
partially refelecting metal films
b. Monochromators :
Gratings
Prism

26. Cross-sectional view of an interference filter

27. Properchoiceofprimaryandsecondaryfilterstoavoidinterferencefromanothersubstance:a)excitationspectra(bothsubstancesfluoresceoversamewavelengthregion,b) fluorescencespectra(bothsubstancesabsorbinsamewavelengthregion).

28. 3) Sample compartment
Cuvettes or cells with area of 1 cm2 .Usually made up of;
Quarz or fused silica ----200 nm~ 800 nm
Glass or plastic ----300 nm~
4) Detectors
Photomultiplier tube

29. 29

30. Types of Fluorescent Molecules
•Experimentallyitisfoundthatfluorescenceisfavouredinrigidmolecules,eg.,phenolphthaleinandfluoresceinarestructurallysimilarasshownbelow.However,fluoresceinshowsafargreaterfluorescencequantumefficiencybecauseofitsrigidity.
• phenolphthalein

31. Types of Fluorescent Molecules
•ItisthoughtthattheextrarigidityimpartedbythebridgingoxygengroupinFluoresceinreducestherateofnonradiativerelaxationsothatemissionbyfluorescencehassufficienttimetooccur.
Fluorescein

32. Application
1.Analysis of medicinal compound:
•A number of drugs can be estimated using fluorimetry.
•Fluorescence is made to produce from non- fluorogenicdrugs by following ways:
(a) Some drugs are capable of exhibiting fluorescence in an appropriate solvent.
e.g. Quinine in 0.1 N Sulfuric acid, Riboflavin in 1% tartaric acid, Aminocrinein 0.1 N HCl.
32

33. (b)Organicandinorganiccompoundscanbemadefluorogenicbychemicalchangesuchasoxidation.E.g.Diphenylhydantoin(phenytoin)isoxidisedbyalkalineKMnO4toformbenzophenonewhichexhibitsflurescence.
(c)Organicandinorganiccompoundsarecomplexedwithsuitablereagentstomakethemfluorogenic.
(d)Whentwoormoredrugsarepresent,eachdrugcanbeestimatedindividuallybyadoptingsuitablemethodlike;
Conversionofacidictoalkalinesolutionorviceversa.
Conversionofionictonon-ioniccompoundorviceversa.
Selectionofwavelengthofexcitationforeachdrug.
Extractionofanyonedrugfromthemixtureandanalysingit.
(e)Preparationoffluorogenicderivativefromnon-fluorogenicdrug.Someoftheexamplesinclude;
Complexofatropinewitheosinissolubleinchloroformandexhibitfluorescence.
Othernon-fluorogenicdrugswhichcanbeanalysedaremorphineandcodeine.
33

34. 2.Analysisofinorganiccompounds
a.EstimationofUraniuminSalts.
b.Somenon-fluorescentinorganicionscanbemadefluorescentbycomplexingitwithnon-fluorescentorganicreagents.Hencetheseelementcanbefluorimetricallyanalysed.
3.FluorescentindicatorsareusefulinfluorimetricdeterminationasthecolorandintensityoffluorescentdependsuponthepHofthesolution.
4.Hydrogenbonding,geometricalisomerism,polymerization,tautomerismandreactionratescanbestudiedfluorimetrically.
5.Fluorimetrycanbeusedforbothqualitativeandquantitativeestimationofsteroids,proteins,plantpigmentsetc
34