Luminol Chemoluminescence

www.wikipedia.orgwww.wikipedia.org 1

Fluorescence or Phosphorescence?

– – * transitions are most favorable for fluorescence.* transitions are most favorable for fluorescence.

is high (100 – 1000 times greater than n – is high (100 – 1000 times greater than n – *)*)

kkFF is also high (absorption and spontaneous emission are related). is also high (absorption and spontaneous emission are related).

Fluorescence lifetime is short (10Fluorescence lifetime is short (10-7-7 – 10 – 10-9-9 s for s for – – * vs. 10* vs. 10-5-5 – 10 – 10-7-7 s for n – s for n – *).*).

Both molecular structure and chemical environment determines if a molecule will or will not luminescence

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

Possible if highly conjugated Possible if highly conjugated due todue to

– – * transitions.* transitions.

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

Nonaromatic Unsaturated HydrocarbonsNonaromatic Unsaturated Hydrocarbons

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Aromatic Hydrocarbons

Ingle and Crouch, Ingle and Crouch, Spectrochemical AnalysisSpectrochemical Analysis

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

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

Most intense fluorescence is found in compounds with aromatic groupsMost intense fluorescence is found in compounds with aromatic groups

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Heterocyclic Aromatics

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

Aromatics containing carbonyl or heteroatoms are more likely to Aromatics containing carbonyl or heteroatoms are more likely to phosphorescephosphoresce

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

Fluorescence is often weaker.Fluorescence is often weaker.

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Aromatic 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..

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Halogen 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..

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Increased 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.

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Rigid Planar Structure

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

Ingle and Crouch, Ingle and Crouch, Spectrochemical Spectrochemical AnalysisAnalysis

FF = 1.0 = 1.0 FF = 0.2 = 0.2

FF = 0.8 = 0.8 not fluorescentnot fluorescent

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Metals

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

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

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

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Solvent Polarity

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

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.htmlhttp://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.html 11

Solvent Polarity

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

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

New York, 1999.New York, 1999.

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

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Temperature

Increasing temperature increases frequency of Increasing temperature increases frequency of collisions (probability of external conversion).collisions (probability of external conversion).

Fluorescence and Phosphorescence

FluorescenceFluorescence Phosphorescence Phosphorescence

… … Labels/TagsLabels/Tags 43994399 13 13

… … DyesDyes 44244424 31 31

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

SciFinder Scholar Citations 2009SciFinder Scholar Citations 2009

www.wikipedia.org14

Fluorescence or Phosphorescence?Publications in Analytical Chemistry

Fluorescence …Fluorescence … Phosphorescence…Phosphorescence…

1084710847 79277927

Advantages:Advantages:

• 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.

Disadvantages:Disadvantages:

• Long timescaleLong timescale

• Less intensity Less intensity

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Shpol’skii Spectroscopy•Analytical potential of fluorescence spectroscopy often limited by unresolved Analytical potential of fluorescence spectroscopy often limited by unresolved band structure (5-50 nm)band structure (5-50 nm)

•homogeneous band broadening homogeneous band broadening – depends directly on radiative deactivation – depends directly on radiative deactivation properties of the excited state (usually 10properties of the excited state (usually 10-3-3 nm) nm)•inhomogeneous band broadening inhomogeneous band broadening – various analyte microenvironments – various analyte microenvironments yields continuum of bands (usually few nm)yields continuum of bands (usually few nm)•Solution: Incorporate molecules in rigid matrix at low temperature to Solution: Incorporate molecules in rigid matrix at low temperature to minimize broadeningminimize broadening

•Result: Very narrow luminescence spectra with each band representing different Result: Very narrow luminescence spectra with each band representing different substitution sites in the host crystalline matrixsubstitution sites in the host crystalline matrix

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Shpol’skii SpectroscopyRequirements:Requirements:1.1. T < 77K with rapid freezing rateT < 77K with rapid freezing rate2.2. Matrix with dimension matchMatrix with dimension match3.3. Low analyte concentrationLow analyte concentration

Instrumentation:Instrumentation:1.1. Xe lamp excitationXe lamp excitation2.2. Cryogenerator with sample cellCryogenerator with sample cell3.3. High resolution monochromator with PMTHigh resolution monochromator with PMT

Analytes:Analytes: polycyclic aromatic compounds in polycyclic aromatic compounds in environmental, toxicological, or geochemical environmental, toxicological, or geochemical systemssystems

17Garrigues and Budzinski, Garrigues and Budzinski, Trends in Analytical ChemistryTrends in Analytical Chemistry, 14 (5), 1995, pages , 14 (5), 1995, pages 231-239.231-239.

18Garrigues and Budzinski, Garrigues and Budzinski, Trends in Analytical ChemistryTrends in Analytical Chemistry, 14 (5), 1995, pages , 14 (5), 1995, pages 231-239.231-239.

Shpol’skii Spectroscopy

Epi-Fluorescence Microscopy

• Light Source - Mercury or xenon lamp (external to reduce thermal effects)Light Source - Mercury or xenon lamp (external to reduce thermal effects)• Dichroic mirror reflects one range of wavelengths and allows another range to Dichroic mirror reflects one range of wavelengths and allows another range to pass.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.html19

Fluorescence 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.html20

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.html21

Fluorescence 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 22

Fluorescence 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 23

Fluorescence Resonance Energy Transfer (FRET)

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Special Fluorescence Techniques

TIRFTIRF

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

LIFLIF

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