Molecular Luminescence Spectrometry

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Chapter 15

• Molecular Luminescence Spectrometry

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Molecular Luminescence Spectrometry

• Fluorescence - A pathway by which an excited atom
  or molecule relaxes to its ground state
  characterized by emission of radiant energy in
  all directions
• excitation brought about by absorption of photon
• Phosphorescence - Similar to fluorescence except
  that phosphorescence typically has significantly
  longer lifetimes
• excitation brought about by absorption of photon
• involves change in electron spin
• Chemluminescence - The emission of energy as
  electromagnetic radiation during a chemical
  reaction
• excited species is formed during the course of a
  chemical reaction

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Molecular Luminescence Spectrometry

• Advantages over absorption technique
• higher sensitivity, detection limits often 1-3
  orders of magnitude lower than those encountered
  in absorption techniques
• large linear concentration ranges
• more specificity
• Disadvantages as compared to absorption
  technique
• because of the higher sensitivity quantitative
  methods often are subject to serious interference
  effects from the matrix ??????
• less widely applicable than absorption techniques
  since many more species absorb UV/Vis than
  exhibit photoluminescence

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Theory of Fluorescence and Phosphorescence

• Resonance fluorescence - atomic species
• Stokes shift - molecular species
• Electron spin
• Pauli exclusion principle
• Singlet/Triplet excited state

change in electron spin
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Theory of Fluorescence and Phosphorescence

• Singlet/Triplet Excited State
• Singlet/triplet transition (or the reverse) is a
  significantly less probable event than the
  corresponding singlet/singlet transition, thus
• average lifetime of an excited triplet state may
  range from 10-4 to several seconds
  (phosphorescence)
• average lifetime of an excited singlet state
  ranges from 10-5 to 10-8 seconds (fluorescence)
• Excitation from the ground state to an excited
  triplet state does NOT occur!!!!!!!!!!!!!!!!!!!!!!
  !!!!!!!!!!!!!!!!!!!!!!!!

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Theory of Fluorescence and Phosphorescence The
Jablonski Diagram
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Theory of Fluorescence and Phosphorescence

• Deactivation processes - the favored route is the
  one that minimizes the lifetime of the excited
  state
• emission of a photon
• fluorescence - 10-8 to 10-5 seconds
• phosphorescence - 10-4 to several seconds
• radiationless processes
• vibrational relaxation - 10-12 seconds or less
  
• internal conversion
• external conversion
• intersystem crossing

Compete with fluorescence and phosphorescence
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Theory of Fluorescence and Phosphorescence

• Radiationless processes
• vibrational relaxation
• because of the short lifetime of vibrational
  relaxation processes compared to fluorescence,
  fluorescence always involves a transition from
  the lowest vibrational level of an excited
  electronic state (one of the reasons for Stokes
  shift)
• internal conversion
• intermolecular process by which a molecule passes
  to a lower energy electronic state without the
  emission of radiation
• particularly efficient when two electronic energy
  levels are sufficiently close for there to be an
  overlap in vibrational energy
• may result in predissociation (the electron moves
  from a higher electronic state to an upper
  vibrational level of a lower electronic state in
  which the vibrational energy is sufficient to
  cause bond rupture)
• (another cause of Stokes shift)

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Theory of Fluorescence and Phosphorescence

• Radiationless processes
• external conversion - collisional quenching
• interaction and energy transfer between the
  excited molecule and the solvent or other solutes
  
• increased solvent viscosity and lower
  temperatures decrease collisional quenching thus
  enhances fluorescence
• intersystem crossing
• spin of electron is reversed
• as with internal conversion, transition is
  enhanced if the vibrational levels of the two
  states overlap
• most common in atoms containing heavy atoms (the
  heavy atom effect)

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Theory of Fluorescence and Phosphorescence

• Variables that Affect Fluorescence and
  Phosphorescence
• Quantum yield kf / (kf ki kec kic
  kpd kd)
• kx rate constants of
• kf fluorescence
• ki internal conversion
• kec external conversion
• kic intersystem crossing
• kpd predissociation
• kd dissociation

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What Electronic properties lead to absorption of
Light?
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What Electronic properties lead to absorption of
Light?

• Look at a simple molecule -- Formaldehyde gt
  H2CO
• Electronic Configuration
• C -- 1s2 2s2 2py1 2px1 2pz0 -----gt1s2
  (3sp2)3 2pz1
• O -- 1s2 2s2 2py2 2px1 2pz1
• C uses 3 sp2 hybrid orbitals to form 3
  s-bonds with O and the 2 H's the
  remaining 2pz orbital forms a p bond with O.
• O has the 2py atomic orbital which is not
  involved in bonding, and it contains a
  non-bonding pair of electrons.

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What Electronic properties lead to absorption of
Light?

• Molecular Orbital Diagram -- only higher energy
  orbitals are shown

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What Electronic properties lead to absorption of
Light?

• Quantum Mechanics
• p ---gt p
• transition is "allowed" and will occur when the
  electric field is parallel to the x axis
• n ---gt p
• transition is "symmetry forbidden" gt
  transition does not induce a dipole change in
  the molecule. This transition does occur
  because of limitations in theory used to predict
  transitions but with very low probability, lt1
  of p ---gt p.

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Quantum Efficiency and Transition Type

• s ---gt s
• transition is seldom observed - too energetic
• most fluorescence occurs from p ---gt p (lifetime
  10-7 to 10-9 s) and p ---gt n (lifetime 10-5 to
  10-7 s) with p ---gt p the most common
  transition because of the shorter lifetimes

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Fluorescence and Structure

• Most unsubstituted aromatic compounds fluoresce
  in solution, with the quantum efficiency
  increasing with the number of rings and their
  condensation
• Simple heterocyclics such as pyridine,
• furan, thiopene, and pyrrole do not
• exhibit fluorescence but fused ring
• structures do.

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Fluorescence and Structure

• Substitution on the benzene ring causes shifts in
  the wavelength of fluorescence maxima and also
  the fluorescence efficiency

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Fluorescence and StructureEffect of pH

• Increased resonance structures lead to enhanced
  fluorescence.
• Relative Fluorescence Intensity
• (relative to benzene with relative fluorescence
  of benzene 10)
• 20
  0

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Fluorescence and StructureEffect of Structural
Rigidity

• Lack of rigidity in a molecule probably causes an
  enhanced internal conversion rate and a
  consequent increase in the likelihood for
  radiationless deactivation.
• Quantum efficiency
• 1.0
  0.2

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Temperature and Solvent Effects

• Quantum efficiency of fluorescence in most
  molecules decreases with increasing temperature
  due to in an increase in the number of collisions
• A decrease in the solvent viscosity has the same
  effect as an increase in temperature for the same
  reason
• The fluorescence of a molecule is also decreased
  by solvents or other solutes containing heavy
  atoms, due to an increase in the rate of triplet
  formation

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Effect of Dissolved Oxygen

• May cause photochemically induced oxidation of
  the fluorescing species
• It also promotes intersystem crossing and and
  conversion of excited molecules to the triplet
  state

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Effect of Concentration

• F Kc
• F is also related to absorbance A abc, thus at
  low concentrations it is linear, but at A gt 0.05
  start to lose linearity
• Two other factors responsible for loss of
  linearity
• self-quenching -collision between excited
  molecules
• self-absorption - wavelength of emission overlaps
  an absorption peak
• All of these factors are greater at higher
  concentrations

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Emission and Excitation Spectra

• Excitation spectra obtained by measuring
  luminescence intensity at a fixed wavelength
  while the excitation wavelength is varied.
• Fluorescence and phosphorescence spectra involve
  excitation at a fixed wavelength while recording
  the emission intensity as a function of
  wavelength
• Phosphorescence generally found at longer
  wavelength than fluorescence. In fact the
  wavelength difference can provide information
  about the energy difference between the triplet
  and singlet state

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Emission and Excitation Spectra

• Fluorescence intensity is dependent on molar
  absorptivity of wavelength of excitation.

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Instrumentation
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Instrumentation

• Sources
• most common source for filter fluorometers is a
  low pressure mercury vapor lamp. It produces
  useful lines for excitation at 254, 302, 313,
  546, 578, 691, and 773 nm.
• High pressure xenon arc lamps are normally used
  with spectrofluorometers.
• Lasers are also used especially for very small
  sample sizes (microbore chromatography) and very
  low concentrations

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Instrumentation

• Filters
• both absorption and interference filters have
  been used in fluorometers
• Monochromators
• most spectrofluorometers are equipped with two
  monochromators (excitation and emission)
• Transducers
• Photomultipliers are the most common and they are
  usually operated in photon counting mode

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Applications

• Many environmental factors exert influences on
  fluorescence properties. The three most common
  are
• Solvent polarity (solvent in this context
  includes interior regions of cells, proteins,
  membranes and other biomolecular structures)
• Proximity and concentrations of quenching species
  
• pH of the aqueous medium - in a cell for instance

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Applications - Solvent Polarity
Fluorescence emission spectra of
2-mercaptoethanol adducts of badan in 1)
toluene 2) chloroform 3) acetonitrile 4)
ethanol 5) methanol 6) water
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Applications - pH
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Applications

• Inorganic Species
• Direct method - involves the formation of a
  fluorescing chelate and the measurement of its
  emission
• Indirect method -involves the diminution of
  fluorescence resulting from the quenching action
  of the substance being determined
• Organic Species
• Most important applications are in the analysis
  of food products, pharmaceuticals, clinical
  samples, and natural products.

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Applications

• Lifetime Measurements
• Increases the selectivity of the method and
  permits the analysis of mixtures that contain
  more than one luminescent species with different
  decay rates

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Applications

• Chemluminescence
• Generally, no wavelength selector is necessary
  since the only source of radiation is the
  chemical reaction between the analyte and reagent
• VERY sensitive, because
• of low noise level
• Very large dynamic range
• Have been employed for
• detection of atmospheric NO,
• O3, sulfur, phosphorus, etc.