Molecular Luminescence Secil Koseoglu 11/13/09

Aequorin: Guiding Star for Scientists

Molecular Luminescencehttp:// Emission of a photon as an excited state molecule returns to a lower state Chemoluminescence Bioluminescence Crystalloluminescence Electroluminescence Radioluminescence Sonoluminescence Thermoluminescence Triboluminescence Photoluminescence FluorescencePhosphorescence

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

 J = 0,  1  v =  1,  2,  3, …  S = 0 (i.e. S  S, T  T) Very Fast  – sec. Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, Selection Rules: Radiative: emission of a photon. Non-radiative: electronic energy is converted to translational, rotational or vibrational energy with no emission. Deactivation Processes Absorption

Vibrational Relaxation Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, Excited molecule rapidly transfers excess vibrational energy to the solvent / medium through collisions. 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. Molecule quickly relaxes into the ground vibrational level in the excited electronic level. Non-radiative process Non-radiative process – sec – sec.

Internal Conversion Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, Transfers into a lower energy electronic state of the same multiplicity without emission of a photon. 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. Favored when there is an overlap of the electronic states’ potential energy curves. Non-radiative process (minimal energy change) Non-radiative process (minimal energy change) ~ s between excited electronic states. ~ s between excited electronic states.

Predissociation & Dissociation Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 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. 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. Dissociation and predissociation are more likely in molecules that absorb at low.

Fluorescence Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, Radiative transition between electronic states with the same multiplicity. Radiative transition between electronic states with the same multiplicity. Almost always a progression from the ground vibrational level of the 1 st excited electronic state. Almost always a progression from the ground vibrational level of the 1 st excited electronic state – sec – sec. Occurs at a lower energy than excitation. Occurs at a lower energy than excitation.

External Conversion Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, Non-radiative transition between electronic states involving transfer of energy to other species (solvent, solutes). Non-radiative transition between electronic states involving transfer of energy to other species (solvent, solutes). Also referred to as quenching. Also referred to as quenching. Modifying conditions to reduce collisions reduces the rate of external conversion. Modifying conditions to reduce collisions reduces the rate of external conversion. Occurs on a comparable time scale as fluorescence. Occurs on a comparable time scale as fluorescence.

Intersystem Crossing Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, Similar to internal conversion except that it occurs between electronic states with different multiplicities. Similar to internal conversion except that it occurs between electronic states with different multiplicities. Slower than internal conversion. Slower than internal conversion. More likely in molecules containing heavy nuclei. More likely in molecules containing heavy nuclei. More likely in the presence of paramagnetic compounds. More likely in the presence of paramagnetic compounds.

Phosphorescence Radiative transition between electronic states of different multiplicities. Radiative transition between electronic states of different multiplicities. Much slower than fluorescence (10 -4 – 10 4 s). Much slower than fluorescence (10 -4 – 10 4 s). Even lower energy than fluorescence. Even lower energy than fluorescence.

Stokes Shift Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, 1998.

Quantum Yield Fraction of absorbed photons that are converted to luminescence, fluorescence or phosphorescence photons. May approach unity in favorable cases.

Fluorescence Quantum Yield All activation and deactivation processes discussed so far can be described using first order rate constants. n S1, n S0 = population densities of S 1 and S 0. k A = rate of absorption k F = rate of fluorescence k nr = rate of non-radiative deactivation processes.

A continuously illuminated sample volume (V) will reach steady-state.

 A,p = k A n S0 V  F,p = k F n S1 V k ec = external conversion (S 1  S 0 ) k ic = internal conversion (S 1  S 0 ) k isc = intersystem crossing (S 1  T 1 ) k pd = predissociation k d = dissociation Fluorescence Quantum Efficiency of a Molecule: typically ~ 10 6 – 10 9 s -1 unitless but describesphotons/molecule

Can put in terms of n S0 :  F,p = n S 0 k A  F V Proportional to the number of fluorophores, the rate of absorption (i.e.  ), the quantum yield and the volume of the sample measured.  F,p =  A,p  F

Are you getting the concept? For a given fluorophore under steady state conditions, excitation of a 1 cm 3 sample volume yields the following first-order rate constants: k f = 5 x 10 7 s -1, k nr = 9 x 10 5 s -1, and k a = 1 x s -1 and an overall rate of fluorescence photon emission of 9.8 x photons/second. What is the molecule number density in the ground electronic state?

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

Phosphorescence Quantum Yield Product of two factors: - fraction of absorbed photons that undergo intersystem crossing. - fraction of molecules in T 1 that phosphoresce. k nr = non-radiative deactivation of S 1. k’ nr = non-radiative deactivation of T 1. Is phosphorescence possible if k P < k F ?

Conditions for Phosphorescence Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, k isc > k F + k ec + k ic + k pd + k d k P > k’ nr

Luminescence Lifetimes Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, Emitted Luminescence will decay with time according to: luminescence radiant power at time t luminescence radiant power at time 0 luminescence lifetime ~10 -5 – s ~10 -4 – 10 s

Quenching Static Quenching Lumophore in ground state and quencher form dark complex. Luminescence is only observed from unbound lumophore. Luminescence lifetime not affected by static quenching. Dynamic Quenching/Collisional Quenching Requires contact between quencher and excited lumophore during collision (temperature and viscosity dependent). Luminescence lifetime drops with increasing quencher concentration. Long-Range Quenching/Förster Quenching Result of dipole-dipole coupling between donor (lumophore) and acceptor (quencher). Rate of energy transfer drops with R -6. Used to assess distances in proteins (good for 2-10 nm).

Are you getting the concept? S. Amemiya et al., Chem. Commun.,1997, Determine the type of quenching being demonstrated in the figures below if the fluorescence lifetime of receptor 1 is unchanged with increasing addition of 3. StaticQuenching

Fluorescence or Phosphorescence?  –  * transitions are most favorable for fluorescence.   is high (100 – 1000 times greater than n –  *)  k F is also high (absorption and spontaneous emission are related).  Fluorescence lifetime is short (10 -7 – s for  –  * vs – s for n –  *).

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

Aromatic Hydrocarbons Ingle and Crouch, Spectrochemical Analysis Low lying  –  * singlet state Fluorescent Phosphorescence is weak because there are no n electrons

Heterocyclic Aromatics Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, Aromatics containing carbonyl or heteroatoms are more likely to phosphoresce n –  * promotes intersystem crossing. Fluorescence is often weaker.

Aromatic Substituents Ingle and Crouch, Spectrochemical Analysis Electron donating groups usually increase  F. Electron donating groups usually increase  F. Electron withdrawing groups usually decrease  F. Electron withdrawing groups usually decrease  F.

Halogen Substituents Ingle and Crouch, Spectrochemical Analysis Internal Heavy Atom Effect Promotes intersystem crossing.  F decreases as MW increases.  P increases as MW increases.  P decreases as MW increases.

Increased Conjugation Ingle and Crouch, Spectrochemical Analysis  F increases as conjugation increases.  P decreases as conjugation increases. Hypsochromic effect and bathochromic shift.

Rigid Planar Structure Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, Ingle and Crouch, Spectrochemical Analysis  F = 1.0  F = 0.2  F = 0.8 not fluorescent

Metals Skoog, Hollar, Nieman, Principles of Instrumental Analysis, Saunders College Publishing, Philadelphia, Metals other than certain lanthanides and actinides (with f-f transitions) are usually not themselves fluorescent. A number of organometallic complexes are fluorescent.