Luminescence is rare in nonaromatic hydrocarbons.Luminescence is rare in nonaromatic hydrocarbons.

Possible if highly Possible if highly conjugated due toconjugated due to – – * transitions.* transitions.

Seyhan Ege, Seyhan Ege, Organic ChemistryOrganic Chemistry, D.C. Heath , D.C. Heath and Company, Lexington, MA, 1989.and Company, Lexington, MA, 1989.

Nonaromatic Unsaturated HydrocarbonsNonaromatic Unsaturated Hydrocarbons

Aromatic HydrocarbonsAromatic Hydrocarbons

Ingle and Crouch, Ingle and Crouch, Spectrochemical AnalysisSpectrochemical Analysis

Low lying Low lying – – * singlet state* singlet stateFluorescentFluorescent

Phosphorescence is weak Phosphorescence is weak because there are no n electronsbecause there are no n electrons

Heterocyclic AromaticsHeterocyclic Aromatics

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

Aromatics containing carbonyl or Aromatics containing carbonyl or heteroatoms are more likely to phosphoresceheteroatoms are more likely to phosphoresce

n – n – * promotes intersystem crossing.* promotes intersystem crossing.

Fluorescence is often weaker.Fluorescence is often weaker.

Aromatic SubstituentsAromatic Substituents

Ingle and Crouch, Ingle and Crouch, Spectrochemical AnalysisSpectrochemical Analysis

• Electron donating groups usually increase Electron donating groups usually increase FF..

• Electron withdrawing groups usually decrease Electron withdrawing groups usually decrease FF..

Halogen SubstituentsHalogen Substituents

Ingle and Crouch, Ingle and Crouch, Spectrochemical AnalysisSpectrochemical Analysis

Internal Heavy Atom EffectInternal Heavy Atom Effect

Promotes intersystem crossing.Promotes intersystem crossing.

FF decreases as MW increases. decreases as MW increases.

PP increases as MW increases. increases as MW increases.

PP decreases as MW increases. decreases as MW increases.

Increased ConjugationIncreased Conjugation

Ingle and Crouch, Ingle and Crouch, Spectrochemical AnalysisSpectrochemical Analysis

FF increases as conjugation increases. increases as conjugation increases.

PP decreases as conjugation increases. decreases as conjugation increases.

Hypsochromic effect and bathochromic shift.Hypsochromic effect and bathochromic shift.

Rigid Planar StructureRigid Planar Structure

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

Ingle and Crouch, Ingle and Crouch, Spectrochemical AnalysisSpectrochemical Analysis

FF = 1.0 = 1.0 FF = 0.2 = 0.2

FF = 0.8 = 0.8 not fluorescentnot fluorescent

MetalsMetals

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

Metals other than certain lanthanides and actinides Metals other than certain lanthanides and actinides (with f-f transitions) are usually not themselves (with f-f transitions) are usually not themselves fluorescent.fluorescent.

A number of organometallic complexes are fluorescent.A number of organometallic complexes are fluorescent.

Fluorescence and PhosphorescenceFluorescence and Phosphorescence

FluorescenceFluorescence Phosphorescence Phosphorescence

… … Labels/TagsLabels/Tags 6064260642 231231

… … DyesDyes 8667386673 412412

Which effect is used more regularly?Which effect is used more regularly?

SciFinder Scholar CitationsSciFinder Scholar Citations

www.wikipedia.org

Fluorescence or Phosphorescence Labels?Fluorescence or Phosphorescence Labels?

Answer from a Commercial ViewAnswer from a Commercial View

http://www.invitrogen.com/http://www.invitrogen.com/

Fluorescence or Phosphorescence?Fluorescence or Phosphorescence?

Commercially Available Phosphorescence LabelsCommercially Available Phosphorescence Labels

Erythrosin derivative Eosin derivativeErythrosin derivative Eosin derivative

http://www.invitrogen.com/http://www.invitrogen.com/

Fluorescence or Phosphorescence?Fluorescence or Phosphorescence?

Publications in Publications in Analytical ChemistryAnalytical Chemistry

Fluorescence …Fluorescence … Phosphorescence…Phosphorescence…

1084710847 79277927

• Phosphorescence is rarer than fluorescence => Higher selectivity.Phosphorescence is rarer than fluorescence => Higher selectivity.

• Phosphorescence: Analysis of aromatic compounds in Phosphorescence: Analysis of aromatic compounds in

environmental samples. environmental samples.

Solvent EffectsSolvent EffectsIncreased viscosity can increase luminescence intensity.Increased viscosity can increase luminescence intensity.

H-bonding and dipole interactions with the solvent contribute H-bonding and dipole interactions with the solvent contribute to the Stokes shift.to the Stokes shift.

Ashutosh Sharma and Stephen Schulman, Ashutosh Sharma and Stephen Schulman, Fluorescence Fluorescence SpectroscopySpectroscopy, John Wiley & Sons, New York, 1999., John Wiley & Sons, New York, 1999.

nonequilibriumnonequilibriumexcited stateexcited state

Solvent PolaritySolvent Polarity

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

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

Solvent PolaritySolvent Polarity

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

TemperatureTemperature

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

Increasing temperature increases kIncreasing temperature increases kss

Joseph Lakowicz, Joseph Lakowicz, Principles of Fluorescence Principles of Fluorescence SpectroscopySpectroscopy, Kluwer Academic / Plenum , Kluwer Academic / Plenum

Publishers, New York, 1999.Publishers, New York, 1999.

Decreasing temperature Decreasing temperature can induce a blue-shift in can induce a blue-shift in

fluorescence.fluorescence.

Shpol’skii SpectroscopyShpol’skii Spectroscopy•Analytical potential of fluorescence spectroscopy Analytical potential of fluorescence spectroscopy

often limited by unresolved band structure (5-50 nm)often limited by unresolved band structure (5-50 nm)•homogeneous band broadeninghomogeneous band broadening – depends – depends

directly on radiative deactivation properties of the directly on radiative deactivation properties of the

excited state (usually 10excited state (usually 10-3-3 nm) nm)•inhomogeneous band broadeninginhomogeneous band broadening – various – various

analyte microenvironments yields continuum of analyte microenvironments yields continuum of

bands (usually few nm)bands (usually few nm)•Solution: Incorporate molecules in rigid matrix at Solution: Incorporate molecules in rigid matrix at

low temperature to minimize broadeninglow temperature to minimize broadening•Result: Very narrow luminescence spectra with Result: Very narrow luminescence spectra with

each band representing different substitution sites in each band representing different substitution sites in

the host crystalline matrixthe host crystalline matrix

Shpol’skii SpectroscopyShpol’skii Spectroscopy

Garrigues and Budzinski, Garrigues and Budzinski, Trends in Analytical ChemistryTrends in Analytical Chemistry, 14 (5), , 14 (5), 1995, pages 231-239.1995, pages 231-239.

Requirements:Requirements:

1.1. T < 77K with rapid freezing rateT < 77K with rapid freezing rate

2.2. Matrix with dimension matchMatrix with dimension match

3.3. Low analyte concentrationLow analyte concentration

Instrumentation:Instrumentation:

1.1. Xe lamp excitationXe lamp excitation

2.2. Cryogenerator with sample cellCryogenerator with sample cell

3.3. High resolution monochromator High resolution monochromator

with PMTwith PMTAnalytes: Analytes: polycyclic aromatic polycyclic aromatic

compounds in environmental, compounds in environmental,

toxicological, or geochemical toxicological, or geochemical

systemssystems

Epi-Fluorescence MicroscopyEpi-Fluorescence Microscopy

• Light Source - Mercury or xenon lamp (external to Light Source - Mercury or xenon lamp (external to

reduce thermal effects)reduce thermal effects)• Dichroic mirror reflects one range of wavelengths Dichroic mirror reflects one range of wavelengths

and allows another range to pass.and allows another range to pass.•Barrier filter eliminates all but fluorescent light.Barrier filter eliminates all but fluorescent light.

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

Fluorescence MicroscopyFluorescence Microscopy

Need 3 filters:Need 3 filters:Exciter FiltersExciter FiltersBarrier FiltersBarrier FiltersDichromatic BeamsplittersDichromatic Beamsplitters

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

Are you getting the concept?Are you getting the concept?

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

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

Fluorescence Microscopy ObjectivesFluorescence Microscopy Objectives

Image intensity is a function of the objective numericalImage intensity is a function of the objective numericalaperture and magnification:aperture and magnification:

2

4

)(

)( mag

NAI obj

Fabricated with low fluorescence glass/quartz with anti-Fabricated with low fluorescence glass/quartz with anti-reflection coatingsreflection coatings

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

Fluorescence Microscopy DetectorsFluorescence Microscopy Detectors

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

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

Special Fluorescence TechniquesSpecial Fluorescence Techniques

TIRFTIRF

http://microscopy.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.htmlhttp://microscopy.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html

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