Chapter 15 Molecular Luminescence Spectrometry

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Chapter 15 Molecular Luminescence Spectrometry
Page 357
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Figure 15-1
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Processes that compete with fluorescence

• 1) vibrational relaxation
• 2) internal conversion
• 3) predissociation
• 4) dissociation
• 5) external conversion
• 6) intersystem crossing
• 7) phosphorescence

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Figure 15-1
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Vibrational relaxation

• much faster than fluorescence
• fluorescence occurs at wavelengths longer than
  absorbed radiation, except were no vibrational
  relaxation can take place
• Seen as l'r and lr
• resonance fluorescence - labsorbed lemitted

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Internal conversion

• processes that allow molecule to drop to lower
  electronic E state without emission of photons
• occurs by overlap of lowest vibrational level in
  a higher vibrational level with an upper
  vibrational level in a lower E electronic state
  which then leads to vibrational relaxation
• can occur between
• 2 or more upper electronic states (S1 and S2 in
  Fig. 15-1)
• lowest excited electronic state and electronic
  ground state (S1 and S0 in Fig. 15-1).
• rate constant kic

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Predissociation

• result of internal conversion when electron moves
  to upper vibrational level in a lower electronic
  state that can cause bond rupture
• rate constant kpd

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Dissociation

• initial absorption promotes electron to upper
  vibrational level in excited electronic state
  that causes immediate dissociation
• rate constant kd

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External Conversion

• E transfer between excited molecule and solvent
  or other solutes
• rate constant kec

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Intersystem Crossing

• spin of electron is reversed (singlet to triplet)
• most common causes
• molecules containing I or Br (heavy atom effect)
  or in solutions containing these atoms (or ions)
• O2 or other paramagnetic species
• rate constant ki

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heavy atom effect on fluorescence of quinine
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Phosphorescence

• Can only occur after intersystem crossing
• is much slower than fluorescence

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Quantum yield

• quantum yield f quanta emitted
  quanta absorbed
• f kf
  kf ki kec kic kpd kd

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Transitions that cause fluorescence

• n to p and p to p
• p to p has best quantum efficiency because
• it has shortest excited state lifetime -
  10-7-10-9s vs. 10-5-10-7 for n to p (larger kf)
• intersystem crossing less likely
• s to s often causes predissociation and
  dissociation

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Other factors that affect fluorescence

• 1) structure
• 2) temperature
• 3) viscosity
• 4) pH

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Structure

• fused ring aromatic is best
• conjugated systems next best
• rigid structure best Thought to reduce internal
  conversion

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Temperature and Viscosity

• lower T leads to higher fluorescence
• higher viscosity leads to higher fluorescence
• both reduce rate of external conversion

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pH

• acidic or basic substituents can cause change in
  fluorescence as pH changes
• protonation or deprotonation of acidic or basic
  functional groups can cause changes in number of
  resonance structures

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Effect of pH change on quinine fluorescence
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Effects of concentration on fluorescence

• Fluorescence is directly proportional to
  concentration at low concentrations but shows
  negative deviation at higher concentrations for 3
  reasons
• 1) selfquenching excited molecules collide and
  deactivate in similar fashion to external
  conversion. Collisions of excited analyte
  molecules are more likely at higher
  concentrations
• 2) self-absorption (inner filter effect) if
  excitation and emission spectra overlap, then
  some of emitted radiation can be reabsorbed by
  ground state analyte molecules.

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Self-absorption
Figure 15-5
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Effects of concentration on fluorescence

• 3) theoretical as seen below, higher order
  terms in equation relating concentration and
  fluorescence cause negative deviations
• PF kfF(P0 P) (1)
• PF power of fluorescence signal
• (P0 P) amount of light absorbed by analyte
• k is an instrumental term composed of two terms
• k f(?)g(l).
• f(?) is a measure of how much of emitted light
  the instrument collects, and g(l) is detector
  response at emission l

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Effects of concentration on fluorescence

• A ebC
• - log (P/P0) ebC
• P/P0 10?ebC fraction of
  light transmitted
• 1 (P/P0) 1 ? 10?ebC fraction of light
  absorbed
• P0 - P P0(1 ? 10?ebC) actual amount
  absorbed (2)

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Effects of concentration on fluorescence

• substituting equation 2 into equation 1
• PF kfFP0(1 ? 10 ?ebC)
• kfFP02.303ebC ? (2.303ebC)2
  (2.303ebC)3?etc
• 1! 2!
  3!
• if 2.303ebC lt 0.05, higher order terms drop out
  and equation reduces to
• PF 2.303ebCkfFP0
• at higher concentrations, higher order terms
  cannot be neglected and they cause negative
  deviations

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differences between PF kfFP0(1 ? 10 ?ebC) and A
ebC

• 1) PF ? P0, so signal can be increased by
  increasing P0.
• Not possible for absorption measurements
  because
• A log(P0/P), and as P0 is increased, P
  increases by same relative amount, so ratio is
  unchanged.
• 2) k term can be increased by more efficient
  collection of light.
• Cannot be done for absorbance measurement.
• 3) Because of 1 and 2 there is no fixed maximum
  fluorescence to which instrument can be set. (A
  instrument is set to T 0).
• A standard (quinine) must be used to compare
  results from different instruments.
• 4) Fluorescence signal is easily amplified.
  Absorbance cannot be increased by amplification.

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Fluorometer and Spectrofluorometer components

• Sources
• 1) low P Hg lamp useful in fluorometers. Emits
  many lines in UV and VIS.
• 2) Xe lamp continuous and more powerful used in
  spectrofluorometers
• 3) lasers not as common but can be very useful

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Fluorometer and Spectrofluorometer components

• Wavelength selectors filters and monochromators
  (new - polychromators)
• Transducers PMTs, PDAs, and CCDs or CIDs
• Cells Pyrex or quartz, no fingerprints
• Cell compartments flat black, baffles to reduce
  scattered light

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Instrument Designs - Fluorometer
Figure 15-6
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Instrument Designs - Spectrofluorometer
Figure 15-7
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Instrument Designs Spectrofluorometer with
Array Detector
Figure 15-8
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Instrument Designs Spectrofluorometer with
Array Detector
Figure 15-8
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Phosphorescence
Very similar instrumentation to fluorescence
except 1) time delay between excitation and
measurement of phosphorescence so no
fluorescence is seen 2) sample measured at
liquid N2 temperature (-196 oC) to minimize
collisional deactivation
Figure 15-3
From Skoog, Holler, and Crouch, Principles of
Instrumental Analysis, 6th ed., p. 417, Thomson
Brooks/Cole, Belmont, CA, 2007.
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Chemiluminescence

• production of light from chemical reaction
• simplest reaction sequence
• A B C D
• C C hn
• Instrumentation?

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Fluorometric Determination of H2O2 in Water

• This method to determine H2O2 is based on
  reaction of scopoletin, a highly fluorescent
  molecule, with H2O2 to produce a non fluorescent
  product.
• scopoletin H2O2 non
  fluorescing product
• lex 365 nm lem 490 nm
• Important aspect of method is that reaction is
  extremely slow unless it is catalyzed.
• Horse radish peroxidase is added to catalyze
  reaction.

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Fluorometric Determination of H2O2 in Water

• This method combines some of the best aspects of
  external calibration curve analysis and standard
  addition analysis.
• Blank measurement is made using the sample, so
  matrix effects are included in blank measurement.
• A scopoletin solution is added to a H2O2 sample
  to begin analysis. Because reaction is so slow,
  no reaction takes place while first fluorescence
  reading is made.
• Matrix effects are taken into account because
  this first reading (and all others) comes from
  scopoletin in the analyte solution.
• After first reading is taken, peroxidase is
  added. Scopoletin and H2O2 react immediately
  causing a decrease in fluorescence signal. Size
  of decrease ? amount of H2O2 in sample.

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Fluorometric Determination of H2O2 in Water

• Calibration curve is constructed by adding known
  amounts of H2O2 to analyte solution and measuring
  further decreases in fluorescence until a
  cumulative change larger than that from original
  reaction is achieved.
• Cumulative changes are plotted versus cumulative
  concentration of added H2O2.
• Concentration of H2O2 that caused D fluorescence
  from initial reaction of scopoletin and H2O2 is
  then determined from calibration curve.

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Fluorometric Determination of H2O2 in Water

• Use 3.00 mL of sample. Add 100.0 mL of pH7.0
  phosphate buffer. This is solution A.
• Zero instrument using solution A.
• Add 250 mL of scopoletin solution to solution A.
  This is solution B.
• Set instrument at maximum signal and record
  reading as Finitial
• Add 20 mL of peroxidase solution to solution B.
  This is solution C.
• Record reading of solution C as Ffinal
• Finitial - Ffinal DFsample ? H2O2 concentration

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H2O2 analysis of rainwater
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H2O2 analysis of rainwater
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Concentration and Error for H2O2 analysis
7.952 0.073 mM H2O2 in 3370 mL What is H2O2
concentration, sc, and RSD in original 3.00 mL
rainwater sample?