Molecular Luminescence Emission of a photon as an excited state molecule returns to a lower state Chemoluminescence Chemoluminescence Bioluminescence Bioluminescence

Molecular LuminescenceMolecular Luminescence

Emission of a photon as an excited state molecule Emission of a photon as an excited state molecule returns to a lower statereturns to a lower state

• ChemoluminescenceChemoluminescence• BioluminescenceBioluminescence• CrystalloluminescenceCrystalloluminescence• ElectroluminescenceElectroluminescence• PhotoluminescencePhotoluminescence• RadioluminescenceRadioluminescence• SonoluminescenceSonoluminescence• ThermoluminescenceThermoluminescence• TriboluminescenceTriboluminescence

http://www.shef.ac.uk/content/1/c6/01/89/68/luminescence.jpghttp://www.shef.ac.uk/content/1/c6/01/89/68/luminescence.jpg

Jablonski DiagramJablonski Diagram

Skoog, Hollar, Nieman, Skoog, Hollar, Nieman, Principles of Instrumental AnalysisPrinciples of Instrumental Analysis, , Saunders College Publishing, Philadelphia, 1998.Saunders College Publishing, Philadelphia, 1998.

AbsorptionAbsorption

J = J = 11

vv = = 1, 1, 2, 2, 3, …3, …

S = 0 (i.e. S S = 0 (i.e. S S, T S, T T) T)

Very Fast Very Fast 10 10-14-14 – 10 – 10-15-15 sec. sec.


Selection Rules:Selection Rules:

Vibrational RelaxationVibrational Relaxation


• Excited molecule rapidly Excited molecule rapidly transfers excess vibrational transfers excess vibrational energy to the solvent / energy to the solvent / medium through collisions.medium through collisions.

• Molecule quickly relaxes Molecule quickly relaxes into the ground vibrational into the ground vibrational level in the excited level in the excited electronic level.electronic level.

• Non-radiative processNon-radiative process

• 1010-11-11 – 10 – 10-10-10 sec. sec.

Internal ConversionInternal Conversion


• Transfers into a lower Transfers into a lower energy electronic state of energy electronic state of the same multiplicity the same multiplicity without emission of a without emission of a photon.photon.

• Favored when there is an Favored when there is an overlap of the electronic overlap of the electronic states’ potential energy states’ potential energy curves.curves.

• Non-radiative process Non-radiative process (minimal energy change)(minimal energy change)

• ~10~10-12-12 s between excited s between excited electronic states.electronic states.

FluorescenceFluorescence


• Radiative transition Radiative transition between electronic states between electronic states with the same multiplicity.with the same multiplicity.

• Almost always a Almost always a progression from the progression from the ground vibrational level of ground vibrational level of the 1the 1stst excited electronic excited electronic state.state.

• 1010-10-10 – 10 – 10-6-6 sec. sec.

• Occurs at a lower energy Occurs at a lower energy than excitation.than excitation.

Stokes ShiftStokes Shift


Relationship Relationship between the shape between the shape of the excitation and of the excitation and fluorescence bands.fluorescence bands.

Ingle and Crouch, Ingle and Crouch, Spectrochemical AnalysisSpectrochemical AnalysisP.R. Callis et. al., Chem. Phys. Lett, 244 (1995), 53-58.P.R. Callis et. al., Chem. Phys. Lett, 244 (1995), 53-58.

shiftshift

Fra

nck-

Con

don

Fa

ctor

Fra

nck-

Con

don

Fa

ctor

External ConversionExternal Conversion


• Non-radiative transition Non-radiative transition between electronic states between electronic states involving transfer of energy involving transfer of energy to other species (solvent, to other species (solvent, solutes).solutes).

• Also referred to as Also referred to as quenching.quenching.

• Modifying conditions to Modifying conditions to reduce collisions reduces reduce collisions reduces the rate of external the rate of external conversion.conversion.

• Occurs on a comparable Occurs on a comparable time scale as fluorescence.time scale as fluorescence.

Intersystem CrossingIntersystem Crossing


• Similar to internal Similar to internal conversion except that it conversion except that it occurs between electronic occurs between electronic states with different states with different multiplicitiesmultiplicities..

• Slower than internal Slower than internal conversion.conversion.

• More likely in molecules More likely in molecules containing heavy nuclei.containing heavy nuclei.

• More likely in the More likely in the presence of paramagnetic presence of paramagnetic compounds.compounds.

Luminol ChemoluminescenceLuminol Chemoluminescence

www.wikipedia.orgwww.wikipedia.org

PhosphorescencePhosphorescence

www.wikipedia.orgwww.wikipedia.org

• Radiative transition between electronic states of Radiative transition between electronic states of different multiplicities.different multiplicities.

• Much slower than fluorescence (10Much slower than fluorescence (10-4-4 – 10 – 1044 s). s).

• Even lower energy than fluorescence.Even lower energy than fluorescence.

DissociationDissociation

Ingle and Crouch, Ingle and Crouch, Spectrochemical AnalysisSpectrochemical Analysis

PredissociationPredissociation


• Occurs if the molecule Occurs if the molecule enters a vibrational level enters a vibrational level above the dissociation limit above the dissociation limit during an internal during an internal conversion.conversion.

• Dissociation and Dissociation and predissociation are more predissociation are more likely in molecules that likely in molecules that absorb at low absorb at low ..

Quantum YieldQuantum Yield

Fraction of absorbed photons that are Fraction of absorbed photons that are converted to luminescence, fluorescence converted to luminescence, fluorescence

or phosphorescence photons.or phosphorescence photons.

May approach unity in favorable cases.May approach unity in favorable cases.

pA,

pL,L

Fluorescence Quantum YieldFluorescence Quantum YieldAll activation and deactivation processes discussed All activation and deactivation processes discussed so far can be described using first order rate so far can be described using first order rate constants.constants.

10

1

SnrFSAS nk k - nk

dt

dn

nnS1S1, n, nS0S0 = population densities of S = population densities of S11 and S and S00..

kkAA = rate of absorption = rate of absorption

kkFF = rate of fluorescence = rate of fluorescence

kknrnr = rate of non-radiative deactivation processes. = rate of non-radiative deactivation processes.

A continuously illuminated sample volume (V) will A continuously illuminated sample volume (V) will reach steady-state.reach steady-state.

0 nk k - nk dt

dn10

1

SnrFSAS

nrF

ASS k k

kn n 0

1

A,p = kAnS0V F,p = kFnS1V

nrF

F

pA,

pF,F k k

k

dpdiscicecF

FF k k k k k k

k

kkecec = external conversion (S = external conversion (S11 S S00))

kkicic = internal conversion (S = internal conversion (S11 S S00))

kkiscisc = intersystem crossing (S = intersystem crossing (S11 T T11))

kkpdpd = predissociation = predissociation

kkdd = dissociation = dissociation

FluorescenceFluorescenceQuantum EfficiencyQuantum Efficiency

of a Molecule:of a Molecule:

typically ~ 10typically ~ 1066 – 10 – 1099 s s-1-1

typically 10typically 1055-10-1077 s s-1-1

typically 10typically 1066-10-1099 s s-1-1

unitless unitless butbutdescribesdescribesphotons/moleculephotons/molecule

Time Scales of ProcessesTime Scales of Processes

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.htmlhttp://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.html

Are you getting the concept?Are you getting the concept?For a given fluorophore under steady state conditions, For a given fluorophore under steady state conditions, excitation of a 1 cmexcitation of a 1 cm33 sample volume yields the following sample volume yields the following first-order rate constants: kfirst-order rate constants: kff = 5 x 10 = 5 x 1077 s s-1-1, k, knrnr = 9 x 10 = 9 x 1055 s s-1-1, ,

and kand kaa = 1 x 10 = 1 x 101414 s s-1-1 and an overall rate of fluorescence and an overall rate of fluorescence

photon emission of 9.8 x 10photon emission of 9.8 x 101919 photons/second. What is photons/second. What is the molecule number density in the ground electronic the molecule number density in the ground electronic state?state?

Phosphorescence Quantum YieldPhosphorescence Quantum Yield


Phosphorescence Quantum YieldPhosphorescence Quantum YieldProduct of two factors:Product of two factors:

- fraction of absorbed photons that undergo fraction of absorbed photons that undergo intersystem crossing.intersystem crossing.

- fraction of molecules in Tfraction of molecules in T11 that phosphoresce. that phosphoresce.

nrP

P

nrF

iscP k' k

k

k k

k

kknrnr = non-radiative deactivation of S = non-radiative deactivation of S11..

k’k’nrnr = non-radiative deactivation of T = non-radiative deactivation of T11..

Is phosphorescence possible if kIs phosphorescence possible if kPP < k < kFF??

Conditions for PhosphorescenceConditions for Phosphorescence


kkiscisc > k > kFF + k + kecec + k + kicic + k + kpdpd + k + kdd

kkPP > k’ > k’nrnr

Luminescence LifetimesLuminescence Lifetimes


Emitted Luminescence will decay with time according to:Emitted Luminescence will decay with time according to:

L

t

LL et

0)(

τ

Φ

(t)Φ

L

0L

L luminescence radiant power at time tluminescence radiant power at time t

luminescence radiant power at time 0luminescence radiant power at time 0

luminescence lifetimeluminescence lifetime

1

1

)'(

)(

nrPP

nrFF

kk

kk

QuenchingQuenchingStatic QuenchingStatic Quenching

Lumophore in ground state and quencher form Lumophore in ground state and quencher form dark dark complexcomplex. Luminescence is only observed from . Luminescence is only observed from unbound lumophore. Luminescence lifetime unbound lumophore. Luminescence lifetime notnot affected by static quenching.affected by static quenching.

Dynamic Quenching/Collisional QuenchingDynamic Quenching/Collisional Quenching

Requires contact between quencher and excited Requires contact between quencher and excited lumophore during collision (temperature and viscosity lumophore during collision (temperature and viscosity dependent). Luminescence lifetime drops with dependent). Luminescence lifetime drops with increasing quencher concentration.increasing quencher concentration.

Long-Range Quenching/Förster QuenchingLong-Range Quenching/Förster Quenching

Result of Result of dipole-dipole couplingdipole-dipole coupling between donor between donor (lumophore) and acceptor (quencher). Rate of energy (lumophore) and acceptor (quencher). Rate of energy transfer drops with Rtransfer drops with R-6-6. Used to assess distances in . Used to assess distances in proteins.proteins.

Fluorescence Resonance Energy Transfer (FRET)Fluorescence Resonance Energy Transfer (FRET)

http://www.olympusfluoview.com/applications/fretintro.htmlhttp://www.olympusfluoview.com/applications/fretintro.html

Are you getting the concept?Are you getting the concept?

S. Amemiya et al., S. Amemiya et al., Chem. Commun.Chem. Commun.,,19971997, 1027., 1027.

Determine the type of quenching being demonstrated Determine the type of quenching being demonstrated in the figures below.in the figures below.

Fluorescence or Phosphorescence?Fluorescence or Phosphorescence?

– – * transitions are most favorable for fluorescence.* transitions are most favorable for fluorescence.

is high (100 – 1000 times greater than n – is high (100 – 1000 times greater than n – *)*)

kkFF is also high (absorption and spontaneous is also high (absorption and spontaneous

emission are related).emission are related).

Fluorescence lifetime is short (10Fluorescence lifetime is short (10-7-7 – 10 – 10-9-9 s for s for – – * vs. 10* vs. 10-5-5 – 10 – 10-7-7 s for n – s for n – *).*).

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Molecular Luminescence Emission of a photon as an excited state molecule returns to a lower state Chemoluminescence Chemoluminescence Bioluminescence Bioluminescence