1. Luminol Chemoluminescence

2. Fluorescence or Phosphorescence?
Both molecular structure and chemical environment determines
if a molecule will or will not luminescence
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 – 10-7 s for n – p*).

3. Nonaromatic Unsaturated Hydrocarbons
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.

4. Aromatic Hydrocarbons
Most intense fluorescence is found in compounds with aromatic groups
Low lying p – p* singlet state
Phosphorescence is weak because there are no n electrons
Ingle and Crouch, Spectrochemical Analysis

5. Heterocyclic Aromatics
Aromatics containing carbonyl or heteroatoms are more likely to phosphoresce
n – p* promotes intersystem crossing.
Fluorescence is often weaker.
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

6. Aromatic Substituents
Electron donating groups usually increase fF.
Electron withdrawing groups usually decrease fF.
Ingle and Crouch, Spectrochemical Analysis

7. Halogen Substituents Internal Heavy Atom Effect
Promotes intersystem crossing.
fF decreases as MW increases.
fP increases as MW increases.
tP decreases as MW increases.
Ingle and Crouch, Spectrochemical Analysis

8. Increased Conjugation
fF increases as conjugation increases.
fP decreases as conjugation increases.
Hypsochromic effect and bathochromic shift.
Ingle and Crouch, Spectrochemical Analysis

9. Rigid Planar Structure
fF = 1.0
fF = 0.2
fF = 0.8
not fluorescent
Ingle and Crouch, Spectrochemical Analysis
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

10. Metals
Metals other than certain lanthanides and actinides (with f-f transitions) are usually not themselves fluorescent.
A number of organometallic complexes are fluorescent.
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

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

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

13. Decreasing temperature can induce a blue-shift in fluorescence.
Increasing temperature increases frequency of collisions (probability of external conversion).
Decreasing temperature can induce a blue-shift in fluorescence.
Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999.

14. Fluorescence and Phosphorescence
Which effect is used more regularly?
SciFinder Scholar Citations 2009
Fluorescence Phosphorescence
… Labels/Tags
… Dyes

15. Fluorescence or Phosphorescence? Publications in Analytical Chemistry
Fluorescence … Phosphorescence…
Advantages:
Phosphorescence is rarer than fluorescence => Higher selectivity.
Phosphorescence: Analysis of aromatic compounds in environmental samples.
Disadvantages:
Long timescale
Less intensity

16. 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

17. Shpol’skii Spectroscopy
Requirements:
T < 77K with rapid freezing rate
Matrix with dimension match
Low analyte concentration
Instrumentation:
Xe lamp excitation
Cryogenerator with sample cell
High resolution monochromator with PMT
Analytes: polycyclic aromatic compounds in environmental, toxicological, or geochemical systems
Garrigues and Budzinski, Trends in Analytical Chemistry, 14 (5), 1995, pages

18. Shpol’skii Spectroscopy
Garrigues and Budzinski, Trends in Analytical Chemistry, 14 (5), 1995, pages

19. Epi-Fluorescence Microscopy
Light Source - Mercury or xenon lamp (external to reduce thermal effects)
Dichroic mirror reflects one range of wavelengths and allows another range to pass.
Barrier filter eliminates all but fluorescent light.

20. Fluorescence Microscopy
Need 3 filters:
Exciter Filters
Barrier Filters
Dichromatic Beamsplitters

21. Are you getting the concept?
You plan to excite catecholamine with the 406 nm line from
a Hg lamp and measure fluorescence emitted at 470 ± 15
nm. Choose the filter cube you would buy to do this.
Sketch the transmission profiles for the three optics.

22. Fluorescence Microscopy Objectives
Image intensity is a function of the objective numerical
aperture and magnification:
Fabricated with low fluorescence glass/quartz with anti-
reflection coatings

23. Fluorescence Microscopy Detectors
No spatial resolution required: PMT or photodiode
Spatial resolution required: CCD

24. Fluorescence Resonance Energy Transfer (FRET)

25. Special Fluorescence Techniques
LIF
TIRF