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1、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*).,Fluorescence or Phosp
2、horescenc,Luminescence is rare in nonaromatic hydrocarbons.,Possible if highly conjugated due top p* transitions.,Seyhan Ege, Organic Chemistry, D.C. Heath and Company, Lexington, MA, 1989.,Nonaromatic Unsaturated Hydrocarbons,Luminescence is rare in nonaro,Aromatic Hydrocarbons,Ingle and Crouch, Sp
3、ectrochemical Analysis,Low lying p p* singlet state,Fluorescent,Phosphorescence is weak because there are no n electrons,Aromatic HydrocarbonsIngle and,Heterocyclic Aromatics,Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 2019.,Aromatics contai
4、ning carbonyl or heteroatoms are more likely to phosphoresce,n p* promotes intersystem crossing.,Fluorescence is often weaker.,Heterocyclic AromaticsSkoog, H,Aromatic Substituents,Ingle and Crouch, Spectrochemical Analysis,Electron donating groups usually increase fF. Electron withdrawing groups usu
5、ally decrease fF.,Aromatic SubstituentsIngle and,Halogen Substituents,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.,Halogen SubstituentsIngle and,Increased Co
6、njugation,Ingle and Crouch, Spectrochemical Analysis,fF increases as conjugation increases.fP decreases as conjugation increases.Hypsochromic effect and bathochromic shift.,Increased ConjugationIngle and,Rigid Planar Structure,Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders Coll
7、ege Publishing, Philadelphia, 2019.,Ingle and Crouch, Spectrochemical Analysis,fF = 1.0,fF = 0.2,fF = 0.8,not fluorescent,Rigid Planar StructureSkoog, H,Metals,Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 2019.,Metals other than certain lanth
8、anides and actinides (with f-f transitions) are usually not themselves fluorescent.,A number of organometallic complexes are fluorescent.,MetalsSkoog, Hollar, Nieman, P,Fluorescence and Phosphorescence,FluorescencePhosphorescence Labels/Tags 172925 Dyes1061256,Which effect is used more regularly?,Sc
9、iFinder Scholar Citations,wikipedia.org,Fluorescence and Phosphorescen,Fluorescence or Phosphorescence Labels?Answer from a Commercial View,invitrogen/,Fluorescence or Phosphorescenc,Fluorescence or Phosphorescence Labels?Answer from a Commercial View,invitrogen/,Fluorescence or Phosphorescenc,Fluor
10、escence or Phosphorescence?Commercially Available Phosphorescence Labels,Erythrosin derivative Eosin derivative,invitrogen/,Fluorescence or Phosphorescenc,Fluorescence or Phosphorescence?Publications in Analytical Chemistry,Fluorescence Phosphorescence1380106 Phosphorescence is rarer than fluorescen
11、ce = Higher selectivity. Phosphorescence: Analysis of aromatic compounds in environmental samples.,Fluorescence or Phosphorescenc,Solvent Effects,Increased viscosity can increase luminescence intensity.,H-bonding and dipole interactions with the solvent contribute to the Stokes shift.,Ashutosh Sharm
12、a and Stephen Schulman, Fluorescence Spectroscopy, John Wiley & Sons, New York, 2019.,Solvent EffectsIncreased visco,Solvent Polarity,Increasing solvent polarity usually causes a red-shift in fluorescence.,micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.html,Solvent PolarityInc
13、reasing sol,Solvent Polarity,Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 2019.,Solvent PolarityJoseph Lakowic,Temperature,Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 2019.,Increa
14、sing temperature increases ks,TemperatureJoseph Lakowicz, Pr,Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 2019.,Decreasing temperature can induce a blue-shift in fluorescence.,Joseph Lakowicz, Principles of,Epi-Fluorescence Microscopy,Light
15、 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.,micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorosources.html,Epi-Fluorescence Microsc
16、opy Li,Fluorescence Microscopy,Need 3 filters:Exciter FiltersBarrier FiltersDichromatic Beamsplitters,microscope.fsu.edu/primer/techniques/fluorescence/filters.html,Fluorescence MicroscopyNeed 3,Are you getting the concept?,You plan to excite catecholamine with the 406 nm line froma Hg lamp and meas
17、ure fluorescence emitted at 470 15nm. Choose the filter cube you would buy to do this.Sketch the transmission profiles for the three optics.,microscope.fsu.edu/primer/techniques/fluorescence/fluorotable3.html,Are you getting the concept?Yo,Fluorescence Microscopy Objectives,Image intensity is a func
18、tion of the objective numericalaperture and magnification:,Fabricated with low fluorescence glass/quartz with anti-reflection coatings,micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.html,Fluorescence Microscopy Object,Fluorescence Microscopy Detectors,No spatial resol
19、ution required: PMT or photodiodeSpatial resolution required: CCD,micro.magnet.fsu.edu/primer/digitalimaging/digitalimagingdetectors.html,Fluorescence Microscopy Detect,Special Fluorescence Techniques,TIRF,microscopy.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html,LIF,Special Fluorescence Technique,
