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..

Dkkdj

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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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