Chapter 15 Molecular Luminescence Spectrometry

Important topics in this chapter: Energy diagram and basic concepts Fluorescence quantum yield Fluorescence instrumentation

Luminescence: Energy diagram and basic concepts 2. The factors affect fluorescence 3. Excitation and emission spectra 4. Instrumentation 5. Applications

15A THEORY OF FLUORESCENCE AND PHOSPHORESCENCE

15A-1 Excited States Producing Fluorescence and Phosphorescence

Electron Spin Singlet/Triplet Excited States

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). Ground Single state Excited Single state Excited triplet 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 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 singlet state (b). If the spins become unpaired, the molecule is in an excited triplet state (c).

Molecular Fluorescence Dkkdj Optical emission from molecules that have been excited to higher energy levels by absorption of electromagnetic radiation.

Energy-Level Diagrams for Photoluminescent Molecules

Photoluminescence Band gap determination. The most common radiative transition in semiconductors is between states in the conduction and valence bands, with the energy difference being known as the band gap. Impurity levels and defect detection. Radiative transitions in semiconductors also involve localized defect levels. The photoluminescence energy associated with these levels can be used to identify specific defects.

Photoluminescence Recombination mechanisms. The return to equilibrium, also known as "recombination," can involve both radiative and nonradiative processes. The amount of photoluminescence and its dependence on the level of photo-excitation and temperature are directly related to the dominant recombination process. Material quality. In general, nonradiative processes are associated with localized defect levels. Material quality can be measured by quantifying the amount of radiative recombination.

FIGURE 15-1 Partial energy-level diagram for a photoluminescent system.

15A-2 Rates of Absorption and Emission

15A-3 Deactivation Processes

Vibration Relaxation Internal Conversion External Conversion Intersystem Crossing Phosphorescence

FIGURE 15-2 Fluorescence excitation and emission spectra for a solution of quinine.

15A-4 Variables Affecting Fluorescence and Phosphorescence

Quantum Yield Transition Types in Fluorescence Quantum Efficiency and Transition Type Fluorescence and Structure

Variables that affect Fluorescence and phosphorescence 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 constant ki: Intersystem crossing constant kec: External conversion constant kic: Internal conversion constant kpd: Predissociation constant kd: Dissociation constant p ® p* transitions: high quantum efficiency

Quantum yield = kf / (kf + ki + kec + kic + kpd + kd) + knr Quantum yield = kf / (kf + ki + kec + kic + kpd + kd) 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 Effect of structural rigidity: Molecules with rigid structures have high fluorescence yield. Nonrigid molecule can undergo low-frequency vibrations. kic

What Affects the fluorescence quantum yield? (1) Excitation l Short l's break bonds increase kpre-dis and kdis rarely observed most common s* ®s p* ® n p* ® p (2) Lifetime of state Transition probability measured by e Large e implies short lifetime p*®p > p*® n (10 -9 -10 -7 s > 10 -7 -10 -5 s)

(3) Structure

(4) Rigidity

TABLE 15-1 Effect of Substitution on the fluorescence of Benzene

Effect of Structural Rigidity

Temperature and Solvent Effects Effect of dissolved oxygen

Effect of pH on Fluorescence

Effect of Concentration on Fluorescence Intensity

15A-5 Emission and Excitation Spectra

FIGURE 15-3 Spectra for phenanthrene: E, excitation; F, fluorescence; P, phosphorescence.

15B INSTRUMENTS FOR MEASURING FLUORESCENCE AND PHOSPHORESCENCE

FIGURE 15-4 Components of a fluorometer or spectrofluorometer. 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

Fluorometer Schematic

15B-1Components of Fluorometers and Spectrofluorometers

FIGURE 15-5 Fluorescence spectra for 1 ppm anthracene in alcohol: (a) excitation spectrum; (b) emission spectrum.

Components of Fluorometers and Spectrofluorometers Sources: A more intense source in needed than the tungsten of hydrogen lamp. Lamps: The most common source for filter fluorometer is a low-pressure mercury vapor lamp equipped with a fused silica window. For spectrofluorometers, a 75 to 450-W high-pressure xenon arc lamp in commonly employed. Lasers: Most commercial spectrofluorometers utilize lamp sources because they are less expensive and less troublesome to use.

Components of Fluorometers and Spectrofluorometers Filters and Monochromators: Both interface and absorption filters have been used in fluorometers for wavelength selection of both the excitation beam and the resulting fluorescence radiation. Most spectrofluorometers are equipped with at least one and sometimes two grating monochromators. Transducers: Photomultiplier tubes are the most common transducers in sensitive fluorescence instruments. Cell and Cell Compartments: Both cylindrical and rectangular cell fabricated of glass or silica are employed for fluorescence measurements.

15B-2 Instrument Designs

Fluorometers Spectrofluorometers

FGIURE 15-6 A typical fluorometer.

FIGURE 15-7 A spectrofluorometer.

Spectrofluormeter Schematic

FIGURE 15-8(a) Three-dimensional spectrofluorometer. (a) Schematic of an optical system for obtaining total luminescence spectra with a CCD detector.

hypothetical compound. FIGURE 15-8(b) Excitation and emission spectra of a hypothetical compound.

FIGURE 15-8(c) Total luminescence spectrum of compound in (b).

Spectrofluorometers Based on Array Detectors Fiber-Optic Pluorescence Sensors Phosphorimeters

Figure 15-9 Schematic of a device for alternately exciting and observing phosphorescence.

15B-3 Instrument Standardization

15C APPLICATIONS OF PHOTOLUMINESCENCE METHODS

15C-1 Fluorometric Determination of Inorganic Species

Cations Forming Fluorescing Chelates Fluorometric Reagents

Fluorescence Sensing sensing is based on changes in fluorescence signal either in intensity or in spectrum. Fluorophore based sensors: Enzyme based sensors: Ion sensors DNA/RNA sensors neurotransmitter sensors environmental sensors

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.

and Biochemical Species 15C-2 Methods for Organic and Biochemical Species

TABLE 15-2 Selected Fluorometric Methods for Inorganic Species

15C-3 phosphorimetric methods:

better selectivity; poorer precision; lower temperature; heavy atom results in strong phosphorescence room 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.

in Liquid Chromatography 15C-4 Fluorescence Detection in Liquid Chromatography

15C-5 Lifetime Measurements

Figure 15-10

15D Chemiluminescence

Measurements of chemiluminescence is simple: 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* + D C* ® C + hv NO + O3 ® NO2* +O2 NO2* ® NO2 + hv Measurements of chemiluminescence is simple: only detector, no excitation necessary

15D-1 The Chemiluminescence Phenomenon

15D-2 Measurement of Chemiluminescence

FIGURE 15-11 Chemiluminescence emission intensity as a function of time after mixing reagents.

15D-3 Analytical Applications of Chemiluminescence

Analysis of Gases Analysis for Inorganic Species in the Liquid Phase Analysis for Organic Species

Questions and Problems 15-4