Luminol Chemoluminescence www.wikipedia.org

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. 10-5 – 10-7 s for n – p*).

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.

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

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.

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

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

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

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.

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.

Solvent Polarity Increasing solvent polarity usually causes a red-shift in fluorescence. http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.html

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

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.

Fluorescence and Phosphorescence Which effect is used more regularly? SciFinder Scholar Citations 2009 Fluorescence Phosphorescence … Labels/Tags 4399 13 … Dyes 4424 31 www.wikipedia.org

Fluorescence or Phosphorescence? Publications in Analytical Chemistry Fluorescence … Phosphorescence… 10847 7927 Advantages: Phosphorescence is rarer than fluorescence => Higher selectivity. Phosphorescence: Analysis of aromatic compounds in environmental samples. Disadvantages: Long timescale Less intensity

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

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 231-239.

Shpol’skii Spectroscopy Garrigues and Budzinski, Trends in Analytical Chemistry, 14 (5), 1995, pages 231-239.

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

Fluorescence Microscopy Need 3 filters: Exciter Filters Barrier Filters Dichromatic Beamsplitters http://microscope.fsu.edu/primer/techniques/fluorescence/filters.html

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. http://microscope.fsu.edu/primer/techniques/fluorescence/fluorotable3.html

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

Fluorescence Microscopy Detectors No spatial resolution required: PMT or photodiode Spatial resolution required: CCD http://micro.magnet.fsu.edu/primer/digitalimaging/digitalimagingdetectors.html

Fluorescence Resonance Energy Transfer (FRET)

Special Fluorescence Techniques LIF TIRF http://microscopy.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html