CHAPTER 15: MOLECULAR LUMINESCENCE

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CHAPTER 15 MOLECULAR LUMINESCENCE
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LUMINESCENCE TECHNIQUES

• Emission of light is used to determine certain
  properties,e e.g.structure and concentration, of
  the emitting species.
• Deactivation processes involved in converting a
  substance from excited state to ground state
• the emission of heat,
• activation of a chemical reaction or
• emission of radiation of the same or a modified
  wavelength.
• Forms of photoluminescence (luminescence after
  absorption) are fluorescence (short lifetime) and
  phosphorescence (long lifetime).
• Approximately 10x more sensitive than absorption
  techniquesppb detection limit
• Limited number of systems that photoluminesce.
• Luminescence observed for simple and complex
  systems and for all three phases.

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Theory

• Atoms e.g. dilute Na (g) the
• 3s ?3p transition occurs by absorption at l
  5895 and 5890 A. with a lifetime ? 10-8 sec,
• the electron returns to the ground state
  isotropically (isotropically) emitting hn with
  the wavelength of emission being the same as the
  wavelength of excitation. resonance fluorescence.
  
• Polyatomic Systems
• Resonance fluorescence observed
• Emission of radiation of longer l (called a
  Stoke's shift) more common.. Most fluorescent
  systems are complex organic compounds with 1 or
  more aromatic functional groups so that the
  commonly observed transitions are
• p p, p n, s s, s s.

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EXCITED STATES

• Multiplicity (number of lines observed when the
  molecule is placed in a magnetic field) is
  related number of unpaired spins in the molecule
  (S). M 2S 1.
• Most molecules have an even number of electrons
  which means that all of their electrons in the
  ground state must be paired
• Singlet state (M 20 1). all electrons in
  ground state paired.
• Doublet state (M 2½ 1 2)a free radical
  (substance that has an odd number of electrons).
  electrons can have 2 orientations in the magnetic
  field-opposed to the field and aligned.
• Triplet state (M 21 1 3), excited state
  in which excited electron spin is flipped so that
  the spins are parallel.
• Singlet state Triplet state
• Diamagnetic Paramagnetic
• Probable Less probable
• Lifetime 10-8-10-13sec Lifetime up to 1 s

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ENERGY LEVEL DIAGRAM

• Let ground , the first excited, second excited
  etc. electronic states be S0, S1, S2 and
  etc..for all of the possible singlet states.
• Triplet states would then be T1, T2 and so on .
• All electronic state has several vibrational and
  rotational states.

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Decay Processes

• Internal conversion Movement of electron from one
  electronic state to another without emission of a
  photon, e.g. S2 S1) lasts about 10?12 sec.
• Predissociation internal conversion electron
  relaxes into a state where energy of that state
  is high enough to rupture the bond.
• Vibrational relaxation (10?10-10?11sec)- Energy
  loss associated with electron movement to lower
  vibrational state without photon emission.
• Intersystem crossing Conversion from singlet
  state to a triplet state. e.g. S1 to T1
• External conversion is a non-radiative process in
  which energy of an excited state is given to
  another molecule (e.g. solvent or other solute
  molecules). Related to the collisional frequency
  of excited species with other molecules in the
  solution. Cooling the solution minimizes this
  effect.

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QUANTUM YIELD

• Only a fraction of the photon absorbed result in
  fluorescence. Fraction called Quantum yield
  (efficiency), F
• Excite molecule in say the S1 state can undergo a
  transition back to the ground state S1 S0
  hn. The emitted photon is the useful
  fluorescence line and takes about 10-6-10-10 sec
  to occur.
• Rate of all processes which involved the
  absorption or emission of a photon can be written
  in terms of a first order rate equation

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KINETICS OF ADSORPTION

• The intensity of the light absorbed is
• .DP Po - PT rate of absorption where
• Po photon flux to sample, PT photon flux out
  of sample.
• At steady state rate of absorption equals rate of
  fluorescence or
• DP (kIC kISC kf kQQ)S1
• where kQ rate constant for quenching process-a
  second order process since the Q is also
  important in determining the relative rate of
  this process.
• Let S1 steady state concentration of S1
  molecules. The rate of fluorescence Pf FDP
  F(kIC kISC kf kQQ)S1 and
• Pf kfS1.
• Combine and rearrange F
• large F means a large kf.
• The lifetime of the fluorescing state is given by
  
• The inverse relationship between the rate
  constant and the lifetime tells us that a process
  having a large rate constant has a short lifetime
  and will have the largest fluorescence intensity.

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FLUORESCENCE INTENSITY VS CONCENTRATION

• Before fluorescence occurs absorption must
  occur. The absorption process given by Beer's
  Law
• where e k/2.303 molar absorptivity. We will
  use this in the development of the fundamental
  equation for fluorescence.
• Earlier we stated that
• Pf rate of fluorescence F(kIC kISC kf
  kQQ)S1 FDP FPo - PT.
• Beers law can be written as
• PT Po10-ebC Poe-2.303ebC.
• Substituting into the fluorescence equation
  gives
• Pf FPo - Poe-2.303ebC FPo1 - e-2.303ebC.
  

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Concentration Dependence 2

• From the mathematics handbook
• ex 1 x
• We substitute this for the exponential terms to
  get the following
• For dilute solutions A ebC is small which will
  make the squared and higher powered terms quite
  small.
• e.g. if A 0.05, then the second term is while
  the first term is 2.3030.05 0.115.
• The fluorescence equation then reduces to
• Pf PoF2.303ebC or Pf KC or a linear
  response in the fluorescence intensity with
  concentration will be observed as long as the A lt
  0.05

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Fluorescence Intensity vs Conc.

• When performing a fluorometric analysis, Pf is
  measured independently of Po so that it is not
  necessary to determine P0 i.e. only one
  measurement of intensity is made.
• Remember that Beers law for absorption requires
  measurement of both P and P0
• In the fluorescence experiment, one can increase
  Po and should expect an increase in Pf. Thus,
  one can increase the sensitivity to the analyte
  by increasing the power of the exciting light.
• In the absorption experiment, one needs the ratio
  of the input and output power. Increasing the
  input power also increases the output power but
  does little to the ratio.
• This makes fluorescent techniques inherently more
  sensitive than absorption techniques. The
  detection limits are 10-8M in fluorimetry they
  are 10-12M.

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F AND TRANSITION TYPE

• In absorption spectrum of organics we can observe
  the following transitions to excited states s
  s n s p p n p.
• Fluorescence p. p .Same orbitals possible.
• .s s, and s n transitions are seldom
  observed however when the l of the transition is
  240 nm(UV) since the energy of the transition
  is often high enough to dissociate the molecule.
  
• Less energetic transitions p p p n
  transitions observed.
• .F, for the p p transition is usually greatest
  since process has shortest average lifetime and
  greatest molar absorptivity.
• Other affects on the quantum yield In our
  equation describing quantum yield
• .F
• All terms except kf must be minimized to obtain a
  large fluorescent signal. Chemical structures
  that minimize one or more of the these rate
  constants increase the quantum yield.
• Structural rigidity Decreases the chances of
  vibrational and rotational de-excitation (which
  we have called IC (internal conversion)..
  Prevents the loss of energy by the internal
  conversion process so that the fluorescent yield
  is higher in rigid molecules.
• Ring structures with alternating single and
  double bonds (conjugation) that are aromatic
  usually best fluorescing compounds, although
  highly conjugated aliphatic compounds may also
  fluoresce.
• Temperature Raising the temperature of a system
  increases the collisional frequency between
  excited molecules and the solvent which increases
  the amount of external conversion.
• Solvent Decrease in solvent viscosity also leads
  to increase in external conversion and a decrease
  in fluorescence intensity.

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PHOTOLUMINESCENT ANALYSIS

• Since many compounds fluoresce at the same l,
  fluorescence cannot be used for qualitative
  analysis.
• Quantitative analysis of a large number of
  organic compounds in particular polycyclic
  molecules with extensive conjugation possible.
• E.g. Vitamin A which has a blue-green
  fluorescence with lmax 500 nm in ETOH.
• Often molecules will be polynuclear aromatics
  such as phenol.
• Inorganic species
• Direct form a fluorescent complex with organic
  species and measure the fluorescent intensity.
  Fluorometric agents usually polyfunctional group
  aromatic compounds.
• Indirect diminution of fluorescence measured
  when the ion is added to a fluorescent solution.
  Reaction stoichiometry between the ion and the
  fluorescent reagent must be known.

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Fluorescence Problem

• 5.00 mL of an unknown zinc solution was placed in
  each of two separatory funnels and 4.00 mL of
  1.10 ppm Zn2 was added to the second solution.
  Each was extracted with three 5 mL aliquots of
  CCl4 containing an excess of 8-hydroxyquinoline.
  The extracts were then diluted and their
  fluorescence measured with a fluorometer. The
  fluorescent intensities were 6.12 for the
  solution containing no added zinc and 11.16 for
  the other solution. Determine the concentration
  of the original zinc solution.
• Strategy
• Determine concentration of final solution
  containing the unknown (Standard Addition
  Method).
• Determine concentration of the original solution.

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INSTRUMENTATION

• Source Hg or Xe arc lamp is used. (continuous
  radiation in the 250-600 nm range is produced).
• Monochromators or filters needed to select both
  wavelength of excitation emission.
• Monochromators are used when dealing with narrow
  absorption or emission peaks while filters may be
  used when peaks are not as narrow.
• When filters are used, one is limited to
  wavelength range that passes through the
  particular filter used.
• Instruments using filters are called fluorometers
  while instrument using monochromators are called
  spectrofluorimeter.
• Cells and Cell compartments cylindrical or
  rectangular (less scattering rectangular cell)
  quartz or glass depending upon the wavelength
  range needed Outlet of the sample cell usually
  90 from the inlet.(minimizes source light at
  detector.
• Detector Phototube or photomultiplier (small
  signals)

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A Fluorometer or Spectrofluorometer
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A Sectrofluorometer