chapter 15 molecular luminescence spectrometry 歐亞書局 important topics in this chapter: energy...
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Chapter 15Chapter 15
Molecular Molecular Luminescence Luminescence SpectrometrySpectrometry
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Important topics in this chapter:
Energy diagram and basic concepts
Fluorescence quantum yield
Fluorescence instrumentation
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Luminescence:
1. Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications
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15A THEORY OF FLUORESCENCE
AND PHOSPHORESCENCE
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15A-1 Excited States Producing
Fluorescence and Phosphorescence
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Electron SpinElectron Spin
Singlet/Triplet Excited States Singlet/Triplet Excited States
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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. The electrons are repelled by permanent magnetic fields.
Paramagnetic: magnetic moment and attracted to a magnetic field (due to unpaired electrons).
GroundSingle state
ExcitedSingle state
Excitedtriplet state
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Electronic spin states of molecules. In (a) the ground electronic state Electronic spin states of molecules. In (a) the ground electronic state is shown. In the lowest energy, or ground, state, the spins are always paired, and is shown. In the lowest energy, or ground, state, the spins are always paired, and the state is said to be a singlet state. In (b) and (c), excited electronic states are the state is said to be a singlet state. In (b) and (c), excited electronic states are shown. If the spins remain paired in the excited state, the molecule is in an excited shown. If the spins remain paired in the excited state, the molecule is in an excited singlet state (b). If the spins become unpaired, the molecule is in an excited triplet singlet state (b). If the spins become unpaired, the molecule is in an excited triplet state (c).state (c).
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Molecular FluorescenceMolecular Fluorescence
Optical emission from Optical emission from molecules that have molecules that have been excited to been excited to higher energy levels higher energy levels by absorption of by absorption of electromagnetic electromagnetic radiationradiation..
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Energy-Level Diagrams forEnergy-Level Diagrams for
Photoluminescent MoleculesPhotoluminescent Molecules
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PhotoluminescencePhotoluminescence
Band gap determination.Band gap determination. The most common radiative The most common radiative transition in semiconductors is between states in the transition in semiconductors is between states in the conduction and valence bands, with the energy conduction and valence bands, with the energy difference being known as the band gap.difference being known as the band gap.
Impurity levels and defect detectionImpurity levels and defect detection .. Radiative Radiative transitions in semiconductors also involve localized transitions in semiconductors also involve localized defect levels. The photoluminescence energy defect levels. The photoluminescence energy associated with these levels can be used to identify associated with these levels can be used to identify specific defects. specific defects.
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PhotoluminescencePhotoluminescence
Recombination mechanisms.Recombination mechanisms. The return to The return to equilibrium, also known as "recombination," can equilibrium, also known as "recombination," can involve both radiative and nonradiative processes. involve both radiative and nonradiative processes. The amount of photoluminescence and its The amount of photoluminescence and its dependence on the level of photo-excitation and dependence on the level of photo-excitation and temperature are directly related to the dominant temperature are directly related to the dominant recombination process.recombination process.
Material quality.Material quality. In general, nonradiative processes In general, nonradiative processes are associated with localized defect levels. Material are associated with localized defect levels. Material quality can be measured by quantifying the amount quality can be measured by quantifying the amount of radiative recombination.of radiative recombination.
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FIGURE 15-1FIGURE 15-1 Partial energy-level diagram for a Partial energy-level diagram for a photoluminescent systemphotoluminescent system.
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15A-215A-2 Rates of Absorption and Rates of Absorption and EmissionEmission
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15A-315A-3 Deactivation Processes Deactivation Processes
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Vibration Relaxation Vibration Relaxation
Internal Conversion Internal Conversion
External ConversionExternal Conversion
Intersystem CrossingIntersystem Crossing
Intersystem Crossing Intersystem Crossing
PhosphorescencePhosphorescence
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FIGURE 15-2FIGURE 15-2 Fluorescence excitation and emission spectra for a solution of quinine.
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15A-4 Variables AffectingVariables Affecting Fluorescence and PhosphorescenceFluorescence and Phosphorescence
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Quantum Yield
Transition Types in Fluorescence
Quantum Efficiency and Transition Type
Fluorescence and Structure
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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: Predissociation constantkd: Dissociation constant
* transitions: high quantum efficiency
Variables that affect Fluorescence and phosphorescence
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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
Quantum yield = kf / (kf + ki + kec + kic + kpd + kd)
= kf
kf + knr
Effect of structural rigidity: Molecules with rigid structureshave high fluorescence yield.
Nonrigid molecule can undergo low-frequency vibrations. kic
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What Affects the fluorescence quantum yield?(1) Excitation
Short l's break bonds increase kpre-dis and kdis
rarely observed most commonn
(2) Lifetime of stateTransition probability measured by e
Large e implies short lifetime
n (10 -9 -10 -7 s > 10 -7 -10 -5 s)
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(3) Structure
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(4) Rigidity
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TABLE 15-1TABLE 15-1 Effect of Substitution on the fluorescence of Benzene
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Effect of Structural Rigidity
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Temperature and Solvent Effects
Effect of dissolved oxygen
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Effect of pH on Fluorescence
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Effect of Concentration on Fluorescence Intensity
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15A-5 Emission and Excitation Spectra
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FIGURE 15-3FIGURE 15-3 Spectra for phenanthrene: E, excitation; F, fluorescence; P, phosphorescence.
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15B INSTRUMENTS FOR MEASURING
FLUORESCENCE AND PHOSPHORESCENCE
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Basic design
• components similar to UV/Vis • spectrofluorometers: observe• both excitation & emission spectra.
Extra features for phosphorescence
• sample cell in cooled Dewar flask with liquid nitrogen• delay between excitation and emission
FIGURE 15-4 Components of a fluorometer or spectrofluorometer.
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Fluorometer SchematicFluorometer Schematic
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15B-1Components of Fluorometers Components of Fluorometers and Spectrofluorometersand Spectrofluorometers
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FIGURE 15-5FIGURE 15-5
Fluorescence spectra for 1
ppm anthracene in alcohol:
(a) excitation spectrum; (b)
emission spectrum.
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Components of Fluorometers and Components of Fluorometers and SpectrofluorometersSpectrofluorometers
Sources:Sources: A more intense source in needed than the A more intense source in needed than the tungsten of hydrogen lamp.tungsten of hydrogen lamp.
Lamps:Lamps: The most common source for filter The most common source for filter fluorometer is a low-pressure mercury vapor lamp fluorometer is a low-pressure mercury vapor lamp equipped with a fused silica window. For equipped with a fused silica window. For spectrofluorometers, a 75 to 450-W high-pressure spectrofluorometers, a 75 to 450-W high-pressure xenon arc lamp in commonly employed.xenon arc lamp in commonly employed.
Lasers:Lasers: Most commercial spectrofluorometers Most commercial spectrofluorometers utilize lamp sources because they are less utilize lamp sources because they are less expensive and less troublesome to useexpensive and less troublesome to use..
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Components of Fluorometers and Components of Fluorometers and SpectrofluorometersSpectrofluorometers
Filters and Monochromators:Filters and Monochromators: Both interface and Both interface and absorption filters have been used in fluorometers absorption filters have been used in fluorometers for wavelength selection of both the excitation beam for wavelength selection of both the excitation beam and the resulting fluorescence radiation. Most and the resulting fluorescence radiation. Most spectrofluorometers are equipped with at least one spectrofluorometers are equipped with at least one and sometimes two grating monochromators.and sometimes two grating monochromators.
Transducers:Transducers: Photomultiplier tubes are the most Photomultiplier tubes are the most common transducers in sensitive fluorescence common transducers in sensitive fluorescence instruments.instruments.
Cell and Cell Compartments:Cell and Cell Compartments: Both cylindrical and Both cylindrical and rectangular cell fabricated of glass or silica are rectangular cell fabricated of glass or silica are employed for fluorescence measurements.employed for fluorescence measurements.
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15B-2 Instrument Designs
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Fluorometers
Spectrofluorometers
歐亞書局FGIURE 15-6 A typical fluorometer.
歐亞書局FIGURE 15-7 A spectrofluorometer.
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Spectrofluormeter Schematic
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FIGURE 15-8(a) Three-dimensional spectrofluorometer. (a) Schematic of an optical system for obtaining total luminescence spectra with a CCD detector.
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FIGURE 15-8(b) Excitation and emission spectra of a
hypothetical compound.
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FIGURE 15-8(c) Total luminescence spectrum of compound in (b).
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Spectrofluorometers Based on Array Detectors
Fiber-Optic Pluorescence Sensors
Phosphorimeters
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Figure 15-9Figure 15-9 Schematic of a device for alternately Schematic of a device for alternately exciting and observing phosphorescenceexciting and observing phosphorescence..
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15B-3 Instrument Standardization
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15C APPLICATIONS OFPHOTOLUMINESCENCE METHODS
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15C-1 Fluorometric Determination
of Inorganic Species
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Cations Forming Fluorescing Chelates
Fluorometric Reagents
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Fluorescence Sensing
sensing is based on changes in fluorescence signaleither in intensity or in spectrum.
Fluorophore based sensors:
Enzyme based sensors:
Ion sensors
DNA/RNA sensors
neurotransmitter sensors
environmental sensors
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Some
fluorometric chelating agents
for metal cations. Alizarin
garnet R can detect Al3+ at
levels as low as 0.007 μg/mL.
Detection of F– with alizarin
garnet R is based on
Fluorescence quenching of
the Al3+ complex. Flavanol
can detect Sn4+ at the 0.1 –
μg/mL level.
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15C-2 Methods for Organic
and Biochemical Species
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TABLE 15-2 Selected Fluorometric Methods for Inorganic Species
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15C-3 phosphorimetric methods:
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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.
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15C-4 Fluorescence Detection
in Liquid Chromatography
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15C-5 Lifetime Measurements
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Figure 15-10
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15D Chemiluminescence
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chemiluminescence 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* +O2
NO2* NO2 + hv
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15D-1 The Chemiluminescence
Phenomenon
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15D-2 Measurement of Chemiluminescence
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FIGURE 15-11 Chemiluminescence emission intensity as a
function of time after mixing reagents.
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15D-3 Analytical Applications
of Chemiluminescence
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Analysis of Gases
Analysis for Inorganic Species
in the Liquid Phase
Analysis for Organic Species
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Questions and ProblemsQuestions and Problems 15-415-4
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