5 Resonance Fluorescence Usually atomicEmitted light has same E as excitation lightSimpler, atomic systems with fewer energy states (vs molecules) undergo resonance fluorescenceNot as widely used in analytical chemistry as non-resonance fluorescenceHg analysis is one exampleExcitation BeamEmission (identical E)
6 Non-resonance Fluorescence Typical of molecular fluorescenceLarge number of excited statesrotationalvibrationaletc..Molecules relax by ‘stepping’ from one state to anotherResulting emitted light “shifts” to lower energieslonger wavelengths = lower energyExcitation BeamEmission (lower E, longer )
8 Energy diagram and basic concepts Fluorescence quantum yield Chapter 15 Molecular LuminescenceImportant topics in this chapter:Energy diagram and basic conceptsFluorescence quantum yieldFluorescence instrumentationHomowork in Chapter 15: 1, 2, 3, 4, 6, 7
9 Luminescence: Energy diagram and basic concepts 2. The factors affect fluorescence3. Excitation and emission spectra4. Instrumentation5. Applications
10 Diamagnetic: no net magnetic field due to spin paring. The Singlet: all electron spins are paired; no energy level splitting occurs when 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. Theelectrons are repelled by permanent magnetic fields.Paramagnetic: magnetic moment and attracted to a magnetic field (due to unpaired electrons).GroundSingle stateExcitedSingle stateExcitedtriplet state
11 Partial energy diagram for a photoluminescent system
12 Deactivation processes for an excited state: Vibrational relaxation: fluorescence always involves atransition from the lowest vibrational states of an excited electronic state; electron can return to any one of the vibrational levels of the ground state; s;Internal conversion: intramolecular processes by which a molecule passes to a lower-energy electronic state without emission of radiation.External conversion: 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 photon is emitted.
14 Comparison of Fluorescence and Phosphorescence Fluorescence Phosphorescencelife time short, < 10-5s long, several secondselectron spin no yesexcited states singlet tripletquantum yield high lowtemperature most temperature low temperature more likelyResonance 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.
15 Luminescence: Energy diagram and basic concepts 2. The factors affect fluorescence3. Excitation and emission spectra4. Instrumentation5. Applications
16 Variables that affect Fluorescence and phosphorescence Quantum yield:the ratio of the number of molecules thatluminescence 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: Predissociation constantkd: Dissociation constantp ® p* transitions: high quantum efficiency
17 Quantum yield can be close to unity if the radiationless kfF =kf+ knrQuantum yield = kf / (kf + ki + kec + kic + kpd + kd)Quantum yield can be close to unity if the radiationlessdecay rate is much smaller the the radiative decay.High quantum yield molecules: rhodamine, fluorescein etcEffect of structural rigidity: Molecules with rigid structureshave high fluorescence yield.Nonrigid molecule can undergo low-frequency vibrations. kic
19 I/I0 = 10-ebc Effect of Concentration on Fluorescence Intensity Power of fluorescence emission FF = K’ (I0 –I)I0 and I are the intensities of excitation lights before and after absorbed by the analytes. K’ is the constantrelated to the quantum yieldI/I0 = 10-ebcF = K’ I0 (1–10-ebc)F = 2.3 K’ I0 ebc, (when ebc<0.05)
20 luminescence in quantitative analysis: inherent sensitivity (usually three orders of magnitude better than absorption 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;
21 t = Luminescence Lifetime: average time the molecule spends in the excited state prior to return to the ground state1t =Kf + Knrdetermines the time available for the fluorophore to interactwith or diffuse in its environment, and hence the informationavailable from its emission.
22 Lifetime measurements: ps or fs lasers used for lifetime measurements;fluorescence lifetime refers to the mean lifetime of theexcited 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 lifetime 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.
23 Luminescence: Energy diagram and basic concepts 2. The factors affect fluorescence3. Excitation and emission spectra4. Instrumentation5. Applications
24 Mirror images of absorption and fluorescence spectra: vibrational levels in the ground and excited states havesimilar energy gaps, thus absorption and fluorescence spectra have mirror images (Fig. 15-1).
25 Figure l.3. Absorption and fluorescence emission spectra of perylene and quinine. Emission 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.Qunnine: two absorption bands: 250 nm and 350 nm;fluorescence at 450 nm.
26 Figure 15-2 Fluorescence excitation and emission spectra for a solution of quinine.
27 Figure Fluorescence spectra for 1 ppm anthracene in alcohol: (a) excitation spectrum; (b) emission spectrum.
28 Figure Spectra for phenanthrene: E, 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 New York: Wiley, Reprinted by permission of John Wiley & Sons, Inc.)
29 Luminescence: Energy diagram and basic concepts 2. The factors affect fluorescence3. Excitation and emission spectra4. Instrumentation5. Applications
30 Components of a fluorometer: sources;wavelength selection: two wavelength selection devices;detectors;sample cell.
31 Figure 15-4 Components of a fluorometer of a spectro-fluorometer.
32 Figure 15-6 A typical fluouometer. (courtesy of Farrand Optical Co Figure A typical fluouometer. (courtesy of Farrand Optical Co., Inc.)
33 Figure 15-7 A spectrofluorometer. (Courtesy of SLM Instruments, Inc Figure A spectrofluorometer. (Courtesy of SLM Instruments, Inc., Urbana, IL.)
34 Figure (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 compound. (c) Total luminescence spectrum of compound in b.
35 Figure 15-9 Schematic of a device for alternately exciting and observing phosphorescence.
36 Luminescence: Energy diagram and basic concepts 2. The factors affect fluorescence3. Excitation and emission spectra4. Instrumentation5. Applications
37 Fluorescence 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
39 phosphorimetric methods: better selectivity;poorer precision; lower temperature;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 phosphorescence.
40 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 + hvNO + O3 ® NO2* +O2NO2* ® NO2 + hvMeasurements of chemiluminescence is simple:only detector, no excitation necessary
41 Figure 15-11 Chemiluminescence emission intensity as a function of time after mixing reagents.
43 Instruments for Measuring Absorption of Light….
44 Fluorescence and Phosphorescence Right angleExcitation BeamEmitted BeamDetector
45 Filter = flurometerPrism and grating =spectroflurometer
46 Fluorescence and Phosphorescence Excited single state S1 or S2Excited triplet statephosphorescenceGround stateFluorescence
47 Factors influencing intensity of fluorescence Concentration of fluorescing species FPresence of other solutespHTemperaturePhotocomposition of sample due to sunlightviscosity
48 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%)