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1、2.Spectrofluorimetry,Dr.Hisham E Abdellatefezzat_,Instruments for Measuring Absorption of Light.,Fluorescence and Phosphorescence,Excitation Beam,Emitted Beam,Detector,Resonance Fluorescence,Resonance FluorescenceUsually atomicEmitted light has same E as excitation lightSimpler,atomic systems with f
2、ewer energy states(vs molecules)undergo resonance fluorescenceNot as widely used in analytical chemistry as non-resonance fluorescenceHg analysis is one example,Excitation Beam,Emission(identical E),Non-resonance Fluorescence,Typical of molecular fluorescenceLarge number of excited statesrotationalv
3、ibrationaletc.Molecules relax by stepping from one state to anotherResulting emitted light“shifts”to lower energies longer wavelengths=lower energy,Excitation Beam,Emission(lower E,longer),Important topics in this chapter:,Energy diagram and basic conceptsFluorescence quantum yieldFluorescence instr
4、umentation,Chapter 15 Molecular Luminescence,Homowork in Chapter 15:1,2,3,4,6,7,Luminescence:Energy diagram and basic concepts2.The factors affect fluorescence3.Excitation and emission spectra4.Instrumentation5.Applications,Singlet:all electron spins are paired;no energy level splitting occurs when
5、the molecule is exposed to a magnetic field;Triplet:the electron spins are unpaired and are parallel;excited triplet state is less energetic than the corresponding singlet state.Diamagnetic:no net magnetic field due to spin paring.The electrons are repelled by permanent magnetic fields.Paramagnetic:
6、magnetic moment and attracted to a magnetic field(due to unpaired electrons).,GroundSingle state,ExcitedSingle state,Excitedtriplet state,Partial energy diagram for a photoluminescent system,Deactivation processes for an excited state:Vibrational relaxation:fluorescence always involves a transition
7、from the lowest vibrational states of an excited electronic state;electron can return to any one of the vibrational levels of the ground state;10-12 s;Internal conversion:intramolecular processes by which a molecule passes to a lower-energy electronic state without emission of radiation.External con
8、version:interaction and energy transfer between the excited molecule and the solvent or other molecules.Intersystem crossing:the spin of an excited electron is reversed and a change in multiplicity of the molecule results.Phosphorescence:an excited triplet state to give radiative emission.emission:a
9、 photon is emitted.,Fluorescence and Phosphorescence,Comparison of Fluorescence and PhosphorescenceFluorescencePhosphorescence life timeshort,10-5slong,several seconds electron spinnoyesexcited statessinglettripletquantum yield high lowtemperaturemost temperature low temperature more likely,Resonanc
10、e fluorescence:absorbed radiation is re-emitted without a change in frequency.Stokes shift:molecular fluorescence bands are shifted to wavelengths that are longer than the resonance line.,Luminescence:Energy diagram and basic concepts2.The factors affect fluorescence3.Excitation and emission spectra
11、4.Instrumentation5.Applications,Quantum yield:the ratio of the number of molecules that luminescence to the total number of excited molecules.f=kf/(kf+ki+kec+kic+kpd+kd)kf:Fluorescence constantki:Intersystem crossing constantkec:External conversion constantkic:Internal conversion constantkpd:Prediss
12、ociation constantkd:Dissociation constantp p*transitions:high quantum efficiency,Variables that affect Fluorescence and phosphorescence,Quantum yield can be close to unity if the radiationless decay rate is much smaller the the radiative decay.High quantum yield molecules:rhodamine,fluorescein etc,Q
13、uantum yield=kf/(kf+ki+kec+kic+kpd+kd),F=,kf,kf,+knr,Effect of structural rigidity:Molecules with rigid structureshave high fluorescence yield.,Nonrigid molecule can undergo low-frequency vibrations.kic,Effect of Concentration on Fluorescence Intensity,F=K(I0 I),Power of fluorescence emission F,I0 a
14、nd I are the intensities of excitation lights before and after absorbed by the analytes.K is the constantrelated to the quantum yield,I/I0=10-ebc,F=K I0(110-ebc),F=2.3 K I0 ebc,(when ebc0.05),luminescence in quantitative analysis:inherent sensitivity(usually three orders of magnitude better than abs
15、orption methods;Better selectivity than absorption spectroscopy;The precision and accuracy of photoluminescence method is usually poorer than spectrophotometer by a factor of two to five.Less widely applicable than absorption spectroscopy;,Luminescence Lifetime:average time the molecule spends in th
16、e excited state prior to return to the ground state,determines the time available for the fluorophore to interact with or diffuse in its environment,and hence the information available from its emission.,t=,1,Kf+Knr,Lifetime measurements:ps or fs lasers used for lifetime measurements;fluorescence li
17、fetime refers to the mean lifetime of the excited state,i.e.,the probability of finding a given molecule that has been excited still in the excited state after time t is exp(-t/t0):I=I0 e(-t/t0)precise measurement of the observed lifetime is important since it can be used to calculate the natural li
18、fetime t0(life time in the absence of nonradiative processes,also called intrinsic lifetime).,For a single exponential decay,63%of the molecules have decayed prior to t=t0.,Luminescence:Energy diagram and basic concepts2.The factors affect fluorescence3.Excitation and emission spectra4.Instrumentati
19、on5.Applications,Mirror images of absorption and fluorescence spectra:vibrational levels in the ground and excited states have similar energy gaps,thus absorption and fluorescence spectra have mirror images(Fig.15-1).,Figure l.3.Absorption and fluorescence emission spectra of perylene and quinine.Em
20、ission spectra cannot be correctly presented on both the wavelength and wavenumber scales.The wavenumber presentation is correct in this instance.Wavelengths are shown for convenience.See Chapter 3.Revised from Ref.5.,Internal conversion:excitation by l1 and l2 produces the same fluorescence l3.Qunn
21、ine:two absorption bands:250 nm and 350 nm;fluorescence at 450 nm.,Figure 15-2 Fluorescence excitation and emission spectra for a solution of quinine.,Figure 15-5 Fluorescence spectra for 1 ppm anthracene in alcohol:(a)excitation spectrum;(b)emission spectrum.,Figure 15-3 Spectra for phenanthrene:E,
22、excitation;F,fluorescence;P,phosphorescence.(From W.R.Seitz,in Treatise on Analytical Chemistry,2nd ed.,P.J.Elving,E.J.Meehan,and I.M.Kolthoff,Eds.,Part I,Vol.7,p.169.New York:Wiley,1981.Reprinted by permission of John Wiley&Sons,Inc.),Luminescence:Energy diagram and basic concepts2.The factors affe
23、ct fluorescence3.Excitation and emission spectra4.Instrumentation5.Applications,Components of a fluorometer:,sources;wavelength selection:two wavelength selection devices;detectors;sample cell.,Figure 15-4 Components of a fluorometer of a spectro-fluorometer.,Figure 15-6 A typical fluouometer.(court
24、esy of Farrand Optical Co.,Inc.),Figure 15-7 A spectrofluorometer.(Courtesy of SLM Instruments,Inc.,Urbana,IL.),Figure 15-8(a)Schematic of an optical system for obtaining a total luminescence spectrum with a two-dimensional charge-coupled device.(b)Excitation and emission spectra of hypothetical com
25、pound.(c)Total luminescence spectrum of compound in b.,Figure 15-9 Schematic of a device for alternately exciting and observing phosphorescence.,Luminescence:Energy diagram and basic concepts2.The factors affect fluorescence3.Excitation and emission spectra4.Instrumentation5.Applications,Fluorescenc
26、e Sensingsensing is based on changes in fluorescence signaleither in intensity or in spectrum.,Fluorophore based sensors:Enzyme based sensors:,Ion sensorsDNA/RNA sensorsneurotransmitter sensorsenvironmental sensors,Ion Sensors,phosphorimetric methods:better selectivity;poorer precision;lower tempera
27、ture;heavy atom results in strong phosphorescenceroom temperature methods:deposit analytes on surface:rigid matrix minimize deactivation of the triplet state by external and internal conversions;Using micelles:micelles increase the proximity between heavy metal ion and the phosphur,thus enhance phos
28、phorescence.,Chemiluminescencechemiluminescence is produced when a chemical reaction yields an electronically excited species,which emits light as it returns to its ground states.A+B C*+DC*C+hv,Measurements of chemiluminescence is simple:only detector,no excitation necessary,NO+O3 NO2*+O2NO2*NO2+hv,
29、Figure 15-11 Chemiluminescence emission intensity as a function of time after mixing reagents.,Preview:Laser Chapter 7Homework:Chapter 7:6,Instruments for Measuring Absorption of Light.,Fluorescence and Phosphorescence,Excitation Beam,Emitted Beam,Detector,Right angle,Filter=flurometerPrism and grat
30、ing=spectroflurometer,Fluorescence and Phosphorescence,Excited single state S1 or S2,Ground state,Excited triplet state,phosphorescence,Fluorescence,Factors influencing intensity of fluorescence,Concentration of fluorescing species FPresence of other solutespHTemperaturePhotocomposition of sample du
31、e to sunlight viscosity,Disadvantages of fluoremetry,Dilute solution are less stableAdsorption on the surface of containerOxidation of fluorescence samplePhotodecompositionQuenching(even traces of non fluorescent can quench a fluorescent one in S1 state)It does not exhibit very high precision or accuracy(2 10%),Difference between fluorometry and spectrophotometry,
