Chapter 27

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Dong-Sun Lee / cat-lab / SWU 2010-Fall Version. Chapter 27. Fluorescence spectrometry. What is luminescence ? Luminescence is the emission of photons from electronically excited state. - PowerPoint PPT Presentation

Text of Chapter 27

  • Chapter 27Fluorescence spectrometryDong-Sun Lee / cat-lab / SWU 2010-Fall Version

  • What is luminescence ?Luminescence is the emission of photons from electronically excited state.Luminescence is divided into two types, depending upon the nature of the ground and the excited states.In a singlet excited state, the electron in the higher energy orbital has the opposite spin orientation as the second electron in the lower orbital. These two electrons are said to be paired. Return to the ground state from an excited singlet state does not require an electron to change its spin orientation.In a triplet state these electrons are unpaired, that is, their spins have the same orientation. A change in spin orientation is needed for a triplet state to return to the singlet ground state.diamagnetic S1paramagnetic T1So

  • Types of luminescence(classification according to the means by which energy is supplied to excite the luminescent molecule)1) Photoluminescence : Molecules are excited by interaction with photons of radiation. Fluorescence : Prompt fluorescence : S1 S0 + h The release of electromagnetic energy is immediate or from the singlet state. Delayed fluorescence : S1 T1 S1 S0 + h This results from two intersystem crossings, first from the singlet to the triplet, then from the triplet to the singlet. Phospholuminescence : T1 S0 + h A delayed release of electromagnetic energy from the triplet state. 2) Chemiluminescence : The excitation energy is obtained from the chemical energy of reaction.3) Bioluminescence : Chemiluminescence from a biological system: firefly, sea pansy, jellyfish, bacteria, protozoa, crustacea.4) Triboluminescence : A release of energy when certain crystals, such as sugar, are broken.5) Cathodoluminescence : A release of energy produced by exposure to cathode rays6) Thermoluminescence : When a material existing in high vibrational energy levels emits energy at a temperature below red heat, after being exposed to small amounts of thermal energy.

  • Jablonski diagram.Fluorescence processA: So + h S1 or S2 Radiation process Molecular fluorescence spectrometry is based on the emission of light by molecules that have become electronically excited subsequent to the absorption of visible(400~700nm), UV(200~400nm), or NIR (700 ~ 1100nm) radiation. Excitation process to the excited state from the ground state is very fast, on the order of 1015 s.VR: vibrational relaxation, non-radiational process, 1011 s ~1010 s.IC : internal conversion, S2 S1 S1 S0 non-radiative process, 1012 s.ST : intersystem crossing, S1 T1F : fluorescence, S1 S0 + h 1010~106 s.P : phosphorescence, T1 S0 + h 104 s ~104 s.

  • Example showing that phosphorescence comes at lower energy than fluorescence from the same molecule. The phosphorescence signal is ~10 times weaker than the fluorescence signal and is only observed when the sample is cooled.

  • Photoluminescence methods. Absorption of incident radiation from an external source (a) causes excitation of the analyte to state 1 or state 2 (b). Excited species can dissipate the excess energy by emission of a photon [luminescence (L)] or by radiationless processes (dashed lines) in (b). Emission is isotropic (a), and the frequencies emitted correspond to the energy differences between levels (c).(b)L

  • Emission and chemiluminescence(bioluminescence) methods. In (a) the addition of thermal, electrical or chemical energy causes nonradiational excitation of the analyte and emission of radiation in all directions (isotropic emission). The energy changes that occur during excitation (dashed lines) or emission (soled lines) are shown in (b). The energies of states 1 and 2 are usually relative to the ground level and often abbreviated E1 and E2, respectively. A typical spectrum is shown in (c).SampleEmitted radiation E Thermal, electrical,or chemical energy(a)10E21 = h21 = hc/21E2 = h2 = hc/2E1 = h1 = hc/12(b)E2 1 21(c)

  • Types of fluorescence and emission processesStokes fluorescence : This is the reemission of less energetic photons, which have a longer wavelength than the absorbed photons. One common cause of Stokes shift is the rapid decay to the lowest vibrational level of S1. Furthermore, fluorophores generally decay to excited vibrational levels of So, resulting in further loss of vibrational energy. In addition to these effects, fluorophores can display further Stokes shifts due to solvent effects and excited state reactions. In gas phase, atoms and molecules do not always show Stokes shifts. Anti-Stokes fluorescence : If thermal energy is added to an excited state or a compound has many highly populated vibrational energy levels, emission at shorter wavelengths than those of absorption occurs. This is often observed in dilute gases at high temperature.Resonance fluorescence : This is the reemission of photons possessing the same energy as the absorbed photons. This type of fluorescence is never observed in solution because of solvent interactions, but it does occur in gases and crystals. It is also the basis of atomic fluorescence.Rayleigh scattering : The emitted light has the same wavelength as the exciting light since the absorbed and emitted photons are of the same energy. Raman scattering : This is a form of inelastic scattering which involve a change in the frequency of the incident radiation. Raman scattering involves the gain or loss of vibrational quantum of energy by molecules.

  • Fluorescence efficiency ; quantum yield of fluorescence

    The ratio of the fluorescence radiant power to the absorbed radiant power where the radiant powers are expressed in photons per second.

    = (luminescene radiant power) / ( absorbed radiant power) = (number of photons emitted) / (number of photons absorbed)

    1 0

    The higher the value of , the greater the fluorescence of a compound. A non-fluorescent molecule is one whose quantum efficiency is zero or so close to zero that thee fluorescence is not measurable. All energy absorbed by such a molecule is rapidly lost by collisional deactivation.

  • Fluorescence lifetimeAnother important property of fluorescing molecules is the lifetime () of the lowest excited singlet state. The lifetime of excited state is defined by the average time the molecule spends in the excited state prior to return to the ground state. Generally, fluorescence lifetimes are near 10 nsec. The quantum yield of fluorescence and are related by = kf / (kf+ kd) = kf where kf is the rates of fluorescence, kd is the radiationless rate of deactivation.Fluorescence lifetime measurement is a valuable technique in the analysis of multicomponent samples containing analytes with overlapping fluorescence bands.

    Joseph R. Lakowicz , Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983, pp 9-10.Stephen G. Schulman , (Alan Townshend Edt.), Encyclopedia of analytical science, Vol. 3, Academic Press, London, pp. 1358-1365.

  • Fluorescence related to concentrationThe fluorescence radiant power F is proportional to the absorbed radiant power. F = (Po P)where = fluorescence efficiency, Po = incident power, P = transmitted powerThe relationship between the absorbed radiant power and concentration can be obtained from Beers law. P/ Po = 10A = 10bC P = Po 10bC F = Po (110bC)When expanded in a power series, this equation yields F = Po [(lnbC)1/ 1! ( lnbC)2 / 2! ( lnbC)3 / 3! (lnbC)4 / 4! } (lnbC)n /n!] If bC is 0.05 or less, only the first term in the series is significant and equation can be written as F = Po (lnbC) = kbCwhere k is a constant equal to Poln. Thus, when the concentrations are very dilute and not over 2% of the incident radiation is absorbed, there is linear relationship between fluorescent power and concentration.When bC is greater than about 1.5, 10bC is much less than 1 and fluorescence depends directly on the incident radiation power. F = Po

  • Po Concentration of fluorescing speciesFluorescenceTheoretical behavior of fluorescence as a function of concentration.

  • Structural factors affecting fluorescence1. Fluorescence is expected in molecules that are aromatic or multiple conjugated double bonds with a high degree of resonance stability.2. Fluorescence is also expected in polycyclic aromatic systems.3. Substituents such as NH3, OH, F, OCH3, NHCH3, and N(CH3)2 groups, often enhance fluorescence.4. On the other hand, these groups decrease or quench fluorescence completely : Cl, Br, I, NHCOCH3, NO2, COOH.5. Molecular rigidity enhances fluorescence. Substances fluoresce more brightly in a glassy state or viscous solution. Formation of chelates with metal ions also promotes fluorescence. However, the introduction of paramagnetic metal ions gives rise to phosphorescence but not fluorescence in metal complexes.6. Changes in the system pH, if it affects the charge status of chromophore, may influence fluorescence.

  • Typical aromatic molecules that do not fluoresce.Typical aromatic molecules that fluoresce.

  • Effect of molecular rigidity on quantum yield. The fluorene molecule is held rigid by the central ring, two benzene rings in biphenyl can rotate to one onother.Effect of rigidity on quantum yield in complexes. Free 8-hydroxyquinoline molecules in solution are easily deactivated through collision with solvent molecules and do not fluoresce. The rigidity of the Zn 8-hydroxyquinoline complex enhances fluorescence.

  • Substitution effects on the fluorescence of benzene.

    Substitue