1. Molecular Luminescence
Seçil Köseoğlu
11/15/10

2. Aequorin: Guiding Star for Scientists
he did not want to give the job to a student who needed to succeed in order to get
his or her doctorate

3. Green Fluorescent Protein
Scientists at the Yerkes National Primate Research Center in Atlanta are using green fluorescent protein to study Huntington's disease, which destroys nervous tissue. In 2008 the researchers infected unfertilized monkey eggs with an HIV-like virus, which changed the eggs' DNA to include the defect that causes Huntington's. The virus also introduced a protein that would make rhesus monkeys fluoresce under ultraviolet light (as pictured)--making it easier to study the effects of the disease on the monkeys' brains.

4. Molecular Luminescence
Emission of a photon as an excited state molecule returns to a lower state
Chemoluminescence
Bioluminescence
Crystalloluminescence
Electroluminescence
Radioluminescence
Sonoluminescence
Thermoluminescence
Triboluminescence
Luminescence is the generation of light without heat
Triboluminescence, generated when bonds in a material are broken when that material is scratched, crushed, or rubbed
Sonoluminescence, from imploding bubbles in a liquid when excited by sound
Photoluminescence
Phosphorescence
Fluorescence

5. Theory of Luminescence
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

6. Deactivation Processes
Absorption
Selection Rules:
DJ = 0, 1
Dv = 1, 2, 3, …
DS = 0 (i.e. S  S, T  T)
Very Fast  – sec.
Triplet
Singlet
Deactivation Processes
very rapid – on the order of the period of oscillation of the electric field of a visible photon
Absorption transitions to triplet states are forbidden although weak absorption is possible in some molecules. The triplet state can also be populated from excited singlet state in a process known as intersystem crossing
Radiative: emission of a photon.
Non-radiative: electronic energy is converted to translational, rotational or vibrational energy with no emission.
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

7. Vibrational Relaxation
Excited molecule rapidly transfers excess vibrational energy to the solvent / medium through collisions.
Molecule quickly relaxes into the ground vibrational level in the excited electronic level.
Non-radiative process
10-11 – sec.
Molecule rapidly relaxes to ground vibrational level in a given electronic state
Usually proceeds in stepwise fashion (delta(v) = 1) in which one vibrational quantum is lost per collision
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

8. Internal Conversion
Transfers into a lower energy electronic state of the same multiplicity without emission of a photon.
Favored when there is an overlap of the electronic states’ potential energy curves.
Non-radiative process (minimal energy change)
~10-12 s between excited electronic states.
occurs when potential energy curves for two electronic states cross such that the lower vibrational levels of the electronic state are ~ the same energy as higher vibrational levels of the lower electronic singlet state.
Ultimately the conversion of excess electronic energy to excess vibrational energy
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

9. Predissociation & Dissociation
Occurs when an electron moves from a higher electronic state to an upper vibrational level of a lower electronic state in which the vibrational energy is enough to cause rupture of a bond.
Dissociation and predissociation are more likely in molecules that absorb at low l.
If a bond is ruptured after internal conversion the process is called predissociation…. both processes most likely in molecule that absorb very short wavelength (high E) light (less than 200 nm usually, more than 140 kcal/mole E)
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

10. Fluorescence
Radiative transition between electronic states with the same multiplicity.
Almost always a progression from the ground vibrational level of the 1st excited electronic state.
10-10 – 10-6 sec.
Occurs at a lower energy than excitation.
For most molecules, the electrons are paired in the ground state so that fluorescence involves a singlet-singlet transitions.
Because internal conversion to S1 and vibrational relaxation are more rapid processes than fluorescence, fluorescence almost always occurs from the ground vibrational state of S1 to various vibrational levels in S0. For this reason, only one fluorescence band is normally observed even if absorption to difference singlet states occurs.
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

11. Ingle and Crouch, Spectrochemical Analysis
Relationship between the shape of the excitation and fluorescence bands.
solid line absorption
dashed line emission
don’t see vibronic structure in solution
Often the fluorescence spectrum is a mirror image of the absorption spectrum. This occurs if the vibrational levels in S0 and S1 are similar. Thus, if the FCF for the v”=- to v’ = 2 absorption transition is largest, the corresponding emission transition from v’ =0 to v”=2 is also the strongest. The wavelength of maximum emission is greater because the E difference between the emission levels is less than that between the absorption levels.
Ingle and Crouch, Spectrochemical Analysis

12. External Conversion
Non-radiative transition between electronic states involving transfer of energy to other species (solvent, solutes).
Also referred to as quenching.
Modifying conditions to reduce collisions reduces the rate of external conversion.
Occurs on a comparable time scale as fluorescence.
In some molecules vibrational levels of the ground state overlaps with the first excited state and deactivation occurs rapidly.
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

13. Intersystem Crossing
Similar to internal conversion except that it occurs between electronic states with different multiplicities.
Slower than internal conversion.
More likely in molecules containing heavy nuclei.
More likely in the presence of paramagnetic compounds.
absorption transitions to triplet states are forbidden by symmetry (delta(S) = 0) although weak absorption is possible in some molecules. Triplet state can also be populated from excited singlet states by a process called intersystem crossing which is a crossover between electronic states similar to internal conversion except that the states have different multiplicities (usually singlet to triplet).
After intersystem crossing, a molecules in the triplet(1) state deactivates by vibrational relaxation to the ground vibrational level of T(1). Then the triplet state deactivates by external conversion or intersystem crossing to the ground singlet state
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

14. Phosphorescence
Radiative transition between electronic states of different multiplicities.
Much slower than fluorescence (10-4 – 104 s).
Even lower energy than fluorescence.
Triplet can also deactivate by emission a photon. The radiational deactivation between electronic states with different multiplicity is phosphorescence
An example of phosphorescence. Europium doped strontium silicate-aluminate oxide powder (cyan pigmented). Shown under visible light, long-wave UV light, and in total darkness.
Phosphorescence is a specific type of photoluminescence related to fluorescence. Unlike fluorescence, a phosphorescent material does not immediately re-emit the radiation it absorbs. The slower time scales of the re-emission are associated with 'forbidden' energy state transitions in quantum mechanics. As these transitions occur less often in certain materials, absorbed radiation may be re-emitted at a lower intensity for up to several hours.
In simpler terms, phosphorescence is a process in which energy absorbed by a substance is released relatively slowly in the form of light. This is the mechanism used for 'glow in the dark' materials which are 'charged' by exposure to light. Unlike the relatively swift reactions in a common fluorescent tube, phosphorescent materials used for these materials absorb the energy and 'store' it for a longer time as the subatomic reactions required to re-emit the light occur less often.
Most photoluminescent events, in which a chemical substrate absorbs and then re-emits a photon of light, are fast, on the order of 10 nanoseconds. However, for light to be absorbed and emitted at these fast time scales, the energy of the photons involved (i.e. the wavelength of the light) must be carefully tuned according to the rules of quantum mechanics to match the available energy states and allowed transitions of the substrate. In the special case of phosphorescence, the absorbed photon energy undergoes an unusual intersystem crossing into an energy state of higher spin multiplicity (see term symbol), usually a triplet state. As a result, the energy can become trapped in the triplet state with only quantum mechanically "forbidden" transitions available to return to the lower energy state. These transistions, although "forbidden", will still occur but are kinetically unfavored and thus progress at significantly slower time scales. Most phosphorescent compounds are still relatively fast emitters, with triplet lifetimes on the order of milliseconds. However, some compounds have triplet lifetimes up to minutes or even hours, allowing these substances to effectively store light energy in the form of very slowly degrading excited electron states. If the phosphorescent quantum yield is high, these substances will release significant amounts of light over long time scales, creating so-called "glow in the dark" materials.
Where S is a singlet and T a triplet whose subscripts denote states (0 is the ground state, and 1 the excited state). Transitions can also occur to higher energy levels, but the first excited state is denoted for simplicity.

15. Stokes Shift
Usually fluorescence appears at longer wavelength than absorption because absorption transitions are to higher excited energy states.
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

16. May approach unity in favorable cases.
Quantum Yield
Fraction of absorbed photons that are converted to luminescence, fluorescence or phosphorescence photons.
May approach unity in favorable cases.
Describes the relative efficiencies of radiative and nonradiative routes of deactivation.
There are several rules that deal with fluorescence. The Kasha–Vavilov rule dictates that the quantum yield of luminescence is independent of the wavelength of exciting radiation.
This is not quite true and is violated severely in many simple molecules. A somewhat more reliable statement, although still with exceptions, would be that the fluorescence spectrum shows very little dependence on the wavelength of exciting radiation.

17. Fluorescence Quantum Yield
All activation and deactivation processes discussed so far can be described using first order rate constants.
Related to the rate of absorption and the rate of deactivation of the first excited singlet state.
Assume that all processes are first order wrt number densities of S0 and S1:
Rate of change of the number density of the S1 state is given by this equation…
where the first term describes the rate of absorption
where the second term describes the rate of deactivation (both fluorescent and nonradiative)
nS1, nS0 = population densities of S1 and S0.
kA = rate of absorption
kF = rate of fluorescence
knr = rate of non-radiative deactivation processes.

18. A continuously illuminated sample volume (V) will reach steady-state.
If the fluorophore of interest is contained in a sample volume V that is fully illuminated with radiation of constant intensity, a steady-state concentration of S1 is rapidly achieved

19. FF,p = kFnS1V FA,p = kAnS0V unitless but describes photons/molecule
Fluorescence
Quantum Efficiency
of a Molecule:
typically ~ 106 – 109 s-1
QY describes fraction of absorbed photons which are converted to fluorescence photons
The rates of photon absorption and fluorescence emission are given by the top two equations.
Plug these into equation on previous slide… see that fluoresence Q.Y. is completely dependent on the rate constants for fluorescence and non-radiative decay
if knr >> kf, S1 is deactived by nonradiative properties before the molecule has a chance to fluoresce (and the QY is small).
can even break knr down into all component processes. magnitude of rate constants depends highly on structure and environment of molecule
k(isc) can be much larger in inorganic systems with high atomic number metal ions, usually 10^9 – 10^12 s-1
kec = external conversion (S1  S0)
kic = internal conversion (S1  S0)
kisc = intersystem crossing (S1  T1)
kpd = predissociation
kd = dissociation

20. FF,p = FA,pfF FF,p = nS0kAfFV Can put in terms of nS0:
the rate of fluorescence photon emission is proportional to the ground-state population of the fluorophone, the rate of absroptions, the fluorescence quantum efficiency and the volume element of the sample illuminated
Proportional to the number of fluorophores, the rate of absorption (i.e. e), the quantum yield and the volume of the sample measured.

21. Are you getting the concept?
For a given fluorophore under steady state conditions, excitation of a 1 cm3 sample volume yields the following first-order rate constants: kf = 5 x 107 s-1, knr = 9 x 105 s-1, and ka = 1 x 1014 s-1 and an overall rate of fluorescence photon emission of 9.8 x 1019 photons/second. What is the molecule number density in the ground electronic state?
the rate of fluorescence photon emission is proportional to the ground-state population of the fluorophone, the rate of absroptions, the fluorescence quantum efficiency and the volume element of the sample illuminated

22. Phosphorescence Quantum Yield
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

23. Phosphorescence Quantum Yield
Product of two factors:
fraction of absorbed photons that undergo intersystem crossing.
fraction of molecules in T1 that phosphoresce.
depends on the rate that the triplet state is populated by intersystem crossing and the rate of deactivation of T1
knr = non-radiative deactivation of S1.
k’nr = non-radiative deactivation of T1.
Is phosphorescence possible if kP < kF?

24. Conditions for Phosphorescence
phosphorescence is favored for molecules and environmental conditions in which intersystem crossing is favorable (k(isc) > KF+Kec+kic). Thus if kisc>kf, phosphorescence is favored over fluorescence
even if intersystem crossing is efficient, phosphorescence is not usually observed becauase nonradiative decay of T1 occurs before phosphorescence occurs (k’nr > kp)
kisc > kF + kec + kic + kpd + kd
kP > k’nr
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

25. Luminescence Lifetimes
Emitted Luminescence will decay with time according to:
luminescence radiant power at time t
luminescence radiant power at time 0
luminescence lifetime
If the excitation source is turned off instantaneously, the concentrations of S1 and T1 (and hence, the luminesence) decays. Because the excited states are often deactivated by first-order processes, the decay in either type of luminesence signal can be described by an exponential
lifetime is defined as the time for the luminescnece signal to decay to 1/e of its initial value
More importantly, the lifetime, τ, is independent of the initial intensity of the emitted light. This can be utilized for making non-intensity based measurements in chemical sensing.
~10-5 – 10-8 s
~10-4 – 10 s
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

26. Fluorescence or Phosphorescence?
p – p* transitions are most favorable for fluorescence.
e is high (100 – 1000 times greater than n – p*)
kF is also high (absorption and spontaneous emission are related).
Fluorescence lifetime is short (10-7 – 10-9 s for p – p* vs – 10-7 s for n – p*).
Hard to predict if a molecule exhibits luminescence but some general rules can be stated.
The nature of the lowest-lying excited singlet (S1) is critical in determining the luminescence behavior of a molecule because fluorescence and intersystem crossing usually occur from this state. In organic molecules, the transitions between S0 and S1 can involve pi to pi(star) or n to pi(star) transition. Similarly, the triplet state T1 can be pi to pi(star) or n to pi(star)

27. Nonaromatic Unsaturated Hydrocarbons
Luminescence is rare in nonaromatic hydrocarbons.
Possible if highly conjugated due to
p – p* transitions.
Seyhan Ege, Organic Chemistry, D.C. Heath and Company, Lexington, MA, 1989.

28. Aromatic Hydrocarbons
many aromatic hydrocarbons are intensely fluorescent because they possess a low-lying pi to pi* transition, low E required, minimal bond disruption
Fluorescent
Low lying p – p* singlet state
Phosphorescence is weak because there are no n electrons
Ingle and Crouch, Spectrochemical Analysis

29. Heterocyclic Aromatics
Aromatics containing carbonyl or heteroatoms are more likely to phosphoresce
increased rate of intersystem crossing decreases fluorescence intensity. If the heteroatom is not part of the aromatic ring, the pi to pi* state is lower than the n to pi* state and the molecule fluoresceces (e.g. tryptophan)
n – p* promotes intersystem crossing.
Fluorescence is often weaker.
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

30. Aromatic Substituents
substituents have most effect on lowest lying excited state… al most all groups that have any effect cause a red-shift (bathochromic)
increase fluorescence QY relative to parent compound
electron withdrawing groups introduce a low-lying n to pi* transition, decreasing fluorescnece QY
Electron donating groups usually increase fF.
Electron withdrawing groups usually decrease fF.
Ingle and Crouch, Spectrochemical Analysis

31. Halogen Substituents Internal Heavy Atom Effect
Promotes intersystem crossing.
fF decreases as MW increases.
fP increases as MW increases.
tP decreases as MW increases.
heavy atoms perturb the electron spins and enhance state mixing. This increases the intersystem crossing from S to T and T back to S as well as T1 to S0 phosphorescence… get decreased fluor QY and increased phos QY.
In this table, the ratio of phos QY/ fluor QY varies from 0.60 to >1000 as the halide changes from fluoride to iodide.
Ingle and Crouch, Spectrochemical Analysis

32. Increased Conjugation
fF increases as conjugation increases.
fP decreases as conjugation increases.
Hypsochromic effect and bathochromic shift.
Ingle and Crouch, Spectrochemical Analysis

33. Rigid Planar Structure
fF = 1.0
fF = 0.2
luminescence is favored in molecules with rigid plan structures because the interaction and conjugation of the pi electron system increases and decreases internal conversion from S1 to S0 as well as external conversion
fF = 0.8
not fluorescent
Ingle and Crouch, Spectrochemical Analysis
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

34. Metals
Metals other than certain lanthanides and actinides (with f-f transitions) are usually not themselves fluorescent.
often the ligand itself is not fluorescent but the complex exhibits fluorescence if the lowest-lying singlet state of the ligand is changed from a n,pi* state to a pi, pi*state… sometimes fluorescence even involves charge transfer reaction with metal d electrons
A number of organometallic complexes are fluorescent.
Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

35. Fluorescence or Phosphorescence? Publications in Analytical Chemistry
Advantages:
Phosphorescence is rarer than fluorescence => Higher selectivity.
Phosphorescence: Analysis of aromatic compounds in environmental samples.
Disadvantages:
Long timescale
Less intensity

36. Solvent Polarity
After the fluorophore has been excited to higher vibrational levels of the first excited singlet state (S(1)), excess vibrational energy is rapidly lost to surrounding solvent molecules as the fluorophore slowly relaxes to the lowest vibrational energy level (occurring in the picosecond time scale). Solvent molecules assist in stabilizing and further lowering the energy level of the excited state by re-orienting (termed solvent relaxation) around the excited fluorophore in a slower process that requires between 10 and 100 picoseconds. This has the effect of reducing the energy separation between the ground and excited states, which results in a red shift (to longer wavelengths) of the fluorescence emission. Increasing the solvent polarity produces a correspondingly larger reduction in the energy level of the excited state, while decreasing the solvent polarity reduces the solvent effect on the excited state energy level. The polarity of the fluorophore also determines the sensitivity of the excited state to solvent effects. Polar and charged fluorophores exhibit a far stronger effect than non-polar fluorophores.
Solvent relaxation effects on fluorescence can result in a dramatic effect on the size of Stokes shifts. For example, the heterocyclic indole moiety of the amino acid tryptophan normally resides on the hydrophobic interior of proteins where the relative polarity of the surrounding medium is low. Upon denaturation of a typical host protein with heat or a chemical agent, the environment of the tryptophan residue is changed from non-polar to highly polar as the indole ring emerges into the surrounding aqueous solution. Fluorescence emission is increased in wavelength from approximately 330 to 365 nanometers, a 35-nanometer shift due to solvent effects. Thus, the emission spectra of both intrinsic and extrinsic fluorescent probes can be employed to probe solvent polarity effects, molecular associations, and complex formation with polar and non-polar small molecules and macromolecules.
Increasing solvent polarity usually causes a red-shift in fluorescence.

37. Solvent Polarity
Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999.

38. Increasing temperature increases ks
Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999.

39. Decreasing temperature can induce a blue-shift in fluorescence.
Joseph Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic / Plenum Publishers, New York, 1999.