Fluorescence, Phosphorescence, & Chemiluminescence A) Introduction 1.) Theory of Fluorescence and Phosphorescence: 10-8 – 10-9s M* M + heat 10-5 to 10-8 s fluorescence 10-4 to 10s phosphorescence 10-14 to 10-15 s - Excitation of e- by absorbance of hn. - Re-emission of hn as e- goes to ground state. • Use hn2 for qualitative and quantitative analysis
Fluorescence, Phosphorescence, & Chemiluminescence A) Introduction 1.) Theory of Fluorescence and Phosphorescence: For UV/Vis need to observe Po and P difference, which limits detection For fluorescence, only observe amount of PL
2.) Fluorescence– ground state to single state and back. Phosphorescence - ground state to triplet state and back. 10-5 to 10-8 s 10-4 to 10 s Spins unpaired net magnetic field Spins paired No net magnetic field Fluorescence Phosphorescence Example of Phosphorescence 0 sec 640 sec 1 sec
3) Jablonski Energy Diagram S2, S1 = Singlet States T1 = Triplet State Numerous vibrational energy levels for each electronic state Resonance Radiation - reemission at same l usually reemission at higher l (lower energy) Forbidden transition: no direct excitation of triplet state because change in multiplicity –selection rules.
4.) Deactivation Processes: a) vibrational relaxation: solvent collisions - vibrational relaxation is efficient and goes to lowest vibrational level of electronic state within 10-12s or less. - significantly shorter life-time then electronically excited state - fluorescence occurs from lowest vibrational level of electronic excited state, but can go to higher vibrational state of ground level. - dissociation: excitation to vibrational state with enough energy to break a bond - predissociation: relaxation to vibrational state with enough energy to break a bond
4.) Deactivation Processes: b) internal conversion: not well understood - crossing of e- to lower electronic state. - efficient since many compounds don’t fluoresce - especially probable if vibrational levels of two electronic states overlap, can lead to predissociation or dissociation.
4.) Deactivation Processes: c) external conversion: deactivation via collision with solvent (collisional quenching) - decrease collision increase fluorescence or phosphorescence ‚decrease temperature and/or increase viscosity ‚decrease concentration of quenching (Q) agent. Quenching of Ru(II) Luminescence by O2
4.) Deactivation Processes: d)intersystem crossing: spin of electron is reversed - change in multiplicity in molecule occurs (singlet to triplet) - enhanced if vibrational levels overlap - more common if molecule contains heavy atoms (I, Br) - more common in presence of paramagnetic species (O2)
5.) Quantum Yield (f): ratio of the number of molecules that luminesce to the total number of excited molecules. - determined by the relative rate constants (kx)of deactivation processes f = kf kf + ki + kec+ kic + kpd + kd f: fluorescence I: intersystem crossing ec: external conversion ic: internal conversion pd: predissociation d: dissociation Increase quantum yield by decreasing factors that promote other processes Fluorescence probes measuring quantity of protein in a cell
6.)Types of Transitions: - seldom occurs from absorbance less than 250 nm ‚200 nm 600 kJ/mol, breaks many bonds - fluorescence not seen with s* s - typically p* p or p* n
7.) Fluorescence & Structure: - usually aromatic compounds ‚low energy of pp* transition ‚ quantum yield increases with number of rings and degree of condensation. ‚fluorescence especially favored for rigid structures < fluorescence increase for chelating agent bound to metal. Examples of fluorescent compounds: quinolineindolefluorene8-hydroxyquinoline
8.) Temperature, Solvent & pH Effects: - decrease temperature increase fluorescence - increase viscosity increase fluorescence - fluorescence is pH dependent for compounds with acidic/basic substituents. ‚more resonance forms stabilize excited state. Fluorescence pH Titration resonance forms of aniline
9.) Effect of Dissolved O2: - increase [O2] decrease fluorescence ‚oxidize compound ‚paramagnetic property increase intersystem crossing (spin flipping) Change in fluorescence as a function of cellular oxygen Am J Physiol Cell Physiol 291: C781–C787, 2006.
B) Effect of Concentration on Fluorescence or Phosphorescence power of fluorescence emission: (F) = K’Po(1 – 10 –ebc) K’ ~ f (quantum yield) Po: power of beam ebc: Beer’s law F depends on absorbance of light and incident intensity (Po) At low concentrations: F = 2.3K’ebcPo deviations at higher concentrations can be attributed to absorbance becoming a significant factor and by self-quenching or self-absorption. Fluorescence of crude oil
C) Fluorescence Spectra Excitation Spectra (a) – measure fluorescence or phosphorescence at a fixed wavelength while varying the excitation wavelength. Emission Spectra (b)– measure fluorescence or phosphorescence over a range of wavelengths using a fixed excitation wavelength. Phosphorescence bands are usually found at longer (>l) then fluorescence because excited triple state is lower energy then excited singlet state.
D) Instrumentation - basic design ‚components similar to UV/Vis ‚spectrofluorometers: observe both excitation & emission spectra. - extra features for phosphorescence ‚ sample cell in cooled Dewar flask with liquid nitrogen ‚ delay between excitation and emission
Fluorometers -simple, rugged, low cost, compact - source beam split into reference and sample beam - reference beam attenuated ~ fluorescence intensity A-1 filter fluorometer
Spectrofluorometer -both excitation and emmision spectra - two grating monochromators - quantitative analysis Perkin-Elmer 204
E) Application of Fluorescence - detect inorganic species by chelating ion 8-Hydroxyquinolineflavanolalizarin garnet Rbenzoin
F) Chemiluminescence - chemical reaction yields an electronically excited species that emits light as it returns to ground state. - relatively new, few examples A + B C* C + hn Examples: • Chemical systems - Luminol (used to detect blood) - phenyl oxalate ester (glow sticks)
2) Biochemical systems - Luciferase (Firefly enzyme) “Glowing” Plants Luciferase gene cloned into plants Luciferin (firefly)