Fluorescence, Phosphorescence, & ChemiluminescenceA) Introduction

1.)Theory of Fluorescence and Phosphorescence:- Excitation of e- by absorbance of hn.- Re-emission of hn as e- goes to ground state. Use hn2 for qualitative and quantitative analysis10-14 to 10-15 s10-5 to 10-8 s fluorescence10-4 to 10s phosphorescence

Fluorescence, Phosphorescence, & ChemiluminescenceA) Introduction

1.)Theory of Fluorescence and Phosphorescence:For UV/Vis need to observe Po and P difference, which limits detectionFor fluorescence, only observe amount of PL

MethodMass detection limit (moles)Concentration detection limit (molar)AdvantagesUV-Vis10-13 to 10-1610-5 to 10-8Universalfluorescence10-15 to 10-1710-7 to 10-9Sensitive

2.)Fluorescence ground state to single state and back.Phosphorescence -ground state to triplet state and back.Spins pairedNo net magnetic fieldSpins unpairednet magnetic field10-5 to 10-8 s10-4 to 10 s

3) Jablonski Energy DiagramS2, S1 = Singlet StatesResonance Radiation - reemission at same lusually reemission at higher l (lower energy)Numerous vibrational energy levels for each electronic stateForbidden transition: no direct excitation of triplet state because change in multiplicity selection rules.T1 = Triplet State

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 dont 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 + kdf: fluorescence I: intersystem crossingec: external conversion ic: internal conversionpd: predissociation d: dissociation

Increase quantum yield by decreasing factors that promote other processesFluorescence 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 p p* 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:quinoline indole fluorene 8-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. resonance forms of anilineFluorescence pH Titration

9.)Effect of Dissolved O2:- increase [O2] decrease fluorescence oxidize compound paramagnetic property increase intersystem crossing (spin flipping)Am J Physiol Cell Physiol 291: C781C787, 2006.Change in fluorescence as a function of cellular oxygen

B) Effect of Concentration on Fluorescence or Phosphorescence

power of fluorescence emission: (F) = KPo(1 10 ebc) K ~ f (quantum yield) Po: power of beam ebc: Beers law

F depends on absorbance of light and incident intensity (Po)

At low concentrations: F = 2.3KebcPo

deviations at higher concentrations can be attributed to absorbance becominga 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 wavelengthwhile 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 intensityA-1 filter fluorometer

Spectrofluorometer- both excitation and emmision spectra- two grating monochromators - quantitative analysisPerkin-Elmer 204

E) Application of Fluorescence- detect inorganic species by chelating ion 8-Hydroxyquinoline flavanol alizarin garnet R benzoin

IonReagentAbsorption (nm)Fluorescence (nm)Sensitivity (mg/ml)InterferenceAl3+Alizarin garnet R4705000.007Be, Co, Cr, Cu, F-,NO3-, Ni, PO4-3, Th, ZrF-Al complex of Alizarin garnet R (quenching)4705000.001Be, Co, Cr, Cu, F-,Fe, Ni,PO4-3, Th, Zr

B4O72-Benzoin3704500.04Be, SbCd2+2-(0-Hydroxyphenyl)-benzoxazole365Blue2NH3Li+8-Hydroxyquinoline3705800.2MgSn4+Flavanol4004700.1F-, PO43-, ZrZn2+Benzoin-green10B, Be, Sb, colored ions

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 + hnExamples:Chemical systems- Luminol (used to detect blood)

- phenyl oxalate ester (glow sticks)

2)Biochemical systems- Luciferase (Firefly enzyme)Luciferin (firefly)Glowing PlantsLuciferase gene cloned into plants