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