1

Phosphorescence Quantum Yield

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

2

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?

3

Conditions for Phosphorescence

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

kisc > kF + kec + kic + kpd + kd

kP > k’nr

4

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

5

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*).

6

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

7

Aromatic Hydrocarbons

Ingle and Crouch, Spectrochemical Analysis

Low lying p – p* singlet state

Fluorescent

Phosphorescence is weak because there are no n electrons

8

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.

9

Aromatic Substituents

Ingle and Crouch, Spectrochemical Analysis

• Electron donating groups usually increase fF.

• Electron withdrawing groups usually decrease fF.

10

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.

11

Increased Conjugation

Ingle and Crouch, Spectrochemical Analysis

fF increases as conjugation increases.

fP decreases as conjugation increases.

Hypsochromic effect and bathochromic shift.

12

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

13

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.

14

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

15

Solvent Polarity

Increasing solvent polarity usually causes a red-shift in fluorescence.

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

16

Solvent Polarity

Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999.

17

Temperature

Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999.

Increasing temperature increases ks

18

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

19

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

22

Luminol Chemoluminescence

www.wikipedia.org

23

Applications of Luminescence-Luminescence Quenching• Sensors• FRET-Fluorescence Microscopy• Epi-fluorescence Microscopy• TIRF• PALM

24

Quenching

Non-radiative energy transfer from excited species to other molecules

S0 S1kA

kF

knr

+ Q

kq

25

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

26

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

27

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!

28

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

29

Fluorescence Microscopy

Need 3 filters:Exciter FiltersBarrier FiltersDichromatic Beamsplitters

http://microscope.fsu.edu/primer/techniques/fluorescence/filters.html

30

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

31

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

32

Fluorescence Microscopy Detectors

No spatial resolution required: PMT or photodiodeSpatial resolution required: CCD

http://micro.magnet.fsu.edu/primer/digitalimaging/digitalimagingdetectors.html