phosphorescence quantum yield skoog, hollar, nieman, principles of instrumental analysis, saunders...
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Phosphorescence Quantum Yield
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.
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Phosphorescence Quantum YieldProduct of two factors:
- fraction of absorbed photons that undergo intersystem crossing.
- fraction of molecules in T1 that phosphoresce.
nrP
P
nrF
iscP k' k
k
k k
k
knr = non-radiative deactivation of S1.
k’nr = non-radiative deactivation of T1.
Is phosphorescence possible if kP < kF?
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Conditions for Phosphorescence
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.
kisc > kF + kec + kic + kpd + kd
kP > k’nr
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Luminescence Lifetimes
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.
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 t
luminescence radiant power at time 0
luminescence lifetime
1
1
)'(
)(
nrPP
nrFF
kk
kk
~10-5 – 10-8 s
~10-4 – 10 s
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Fluorescence or Phosphorescence?
p – p* transitions are most favorable for fluorescence.
e is high (100 – 1000 times greater than n – p*)
kF is also high (absorption and spontaneous emission are related).
Fluorescence lifetime is short (10-7 – 10-9 s for p – p* vs. 10-5 – 10-7 s for n – p*).
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Luminescence is rare in nonaromatic hydrocarbons.
Possible if highly conjugated due to
p – p* transitions.
Seyhan Ege, Organic Chemistry, D.C. Heath and Company, Lexington, MA, 1989.
Nonaromatic Unsaturated Hydrocarbons
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Aromatic Hydrocarbons
Ingle and Crouch, Spectrochemical Analysis
Low lying p – p* singlet state
Fluorescent
Phosphorescence is weak because there are no n electrons
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Heterocyclic Aromatics
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.
Aromatics containing carbonyl or heteroatoms are more likely to phosphoresce
n – p* promotes intersystem crossing.
Fluorescence is often weaker.
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Aromatic Substituents
Ingle and Crouch, Spectrochemical Analysis
• Electron donating groups usually increase fF.
• Electron withdrawing groups usually decrease fF.
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Halogen Substituent
Ingle and Crouch, Spectrochemical Analysis
Internal Heavy Atom Effect
Promotes intersystem crossing.
fF decreases as MW increases.
fP increases as MW increases.
tP decreases as MW increases.
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Increased Conjugation
Ingle and Crouch, Spectrochemical Analysis
fF increases as conjugation increases.
fP decreases as conjugation increases.
Hypsochromic effect and bathochromic shift.
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Rigid Planar Structure
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.
Ingle and Crouch, Spectrochemical Analysis
fF = 1.0 fF = 0.2
fF = 0.8 not fluorescent
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Metals
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.
Metals other than certain lanthanides and actinides (with f-f transitions) are usually not themselves fluorescent.
A number of organometallic complexes are fluorescent.
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Fluorescence or Phosphorescence?
Advantages:• Phosphorescence is rarer than fluorescence => Higher selectivity.• Phosphorescence: Analysis of aromatic compounds in
environmental samples.
Disadvantages:• Long timescale• Less intensity
Publications in Analytical ChemistryFluorescence Phosphorescence 1564 34
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Solvent Polarity
Increasing solvent polarity usually causes a red-shift in fluorescence.
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.html
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Solvent Polarity
Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999.
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Temperature
Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999.
Increasing temperature increases ks
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Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers,
New York, 1999.
Decreasing temperature can induce a blue-shift in fluorescence.
Shpol’skii Spectroscopy•Analytical potential of fluorescence spectroscopy often limited by unresolved band structure (5-50 nm)
• homogeneous band broadening – depends directly on radiative deactivation properties of the excited state (usually 10-3 nm)
• inhomogeneous band broadening – various analyte microenvironments yields continuum of bands (usually few nm)
• Solution: Incorporate molecules in rigid matrix at low temperature to minimize broadening
•Result: Very narrow luminescence spectra with each band representing different substitution sites in the host crystalline matrix
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Shpol’skii SpectroscopyRequirements:1. T < 77K with rapid freezing rate2. Matrix with dimension match3. Low analyte concentration
Instrumentation:1. Xe lamp excitation2. Cryogenerator with sample cell3. High resolution monochromator with PMT
Analytes: polycyclic aromatic compounds in environmental, toxicological, or geochemical systems
20Garrigues and Budzinski, Trends in Analytical Chemistry, 14 (5), 1995, pages 231-239.
21Garrigues and Budzinski, Trends in Analytical Chemistry, 14 (5), 1995, pages 231-239.
Shpol’skii Spectroscopy
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Luminol Chemoluminescence
www.wikipedia.org
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Applications of Luminescence-Luminescence Quenching• Sensors• FRET-Fluorescence Microscopy• Epi-fluorescence Microscopy• TIRF• PALM
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Quenching
Non-radiative energy transfer from excited species to other molecules
S0 S1kA
kF
knr
+ Q
kq
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Quantum Yield and Quenching
S0 S1kA
kF
knr
+ Q
kq
Qqnk
nrF
F
pA,
pF,F k k
k
Show that quantum yield in the presence of a quencher is:nrF
F
pA,
pF,F k k
k
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Dynamic Quenching/Collisional QuenchingRequires contact between quencher and excited lumophore during collision (temperature and viscosity dependent). Luminescence lifetime drops with increasing quencher concentration.
QqnrF
QqnrF
f
of nK
kk
nkkk
1
Since fluorescence emission is directly proportional to quantum yield:
QqnKF
F10
Stern-Volmer Equation
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Static QuenchingLumophore in ground state and quencher form dark complex. Luminescence is only observed from unbound lumophore. Luminescence lifetime not affected by static quenching.
Dopamine Sensor!
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Long-Range Quenching/Förster QuenchingResult of dipole-dipole coupling between donor (lumophore) and acceptor (quencher). Rate of energy transfer drops with R-6. Used to assess distances in proteins (good for 2-10 nm).
Förster/Fluorescence Resonance Energy Transfer
Single DNA molecules with molecular Beacons
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Fluorescence Microscopy
Need 3 filters:Exciter FiltersBarrier FiltersDichromatic Beamsplitters
http://microscope.fsu.edu/primer/techniques/fluorescence/filters.html
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Are you getting the concept?You plan to excite catecholamine with the 406 nm line froma Hg lamp and measure fluorescence emitted at 470 ± 15nm. Choose the filter cube you would buy to do this.Sketch the transmission profiles for the three optics.
http://microscope.fsu.edu/primer/techniques/fluorescence/fluorotable3.html
U-MNV
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Fluorescence Microscopy Objectives
Image intensity is a function of the objective numericalaperture and magnification:
2
4
)(
)( mag
NAI obj
Fabricated with low fluorescence glass/quartz with anti-reflection coatings
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.html
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Fluorescence Microscopy Detectors
No spatial resolution required: PMT or photodiodeSpatial resolution required: CCD
http://micro.magnet.fsu.edu/primer/digitalimaging/digitalimagingdetectors.html