Fluorescence, Phosphorescence, & Chemiluminescence

A) Introduction

1.) Theory of Fluorescence and Phosphorescence:

- Excitation of e- by absorbance of h.- Re-emission of hv as e- goes to ground state.- Use h2 for qualitative and quantitative analysis

10-14 to 10-15 s

10-5 to 10-8 s fluorescence10-4 to 10s phosphorescence

Method Mass detection limit (moles)

Concentration detection limit (molar)

Advantages

UV-Vis 10-13 to 10-16 10-5 to 10-8 Universal

fluorescence 10-15 to 10-17 10-7 to 10-9 Sensitive

For UV/Vis need to observe Po and P difference, which limits detection

10-8 – 10-9s

M* M + heat

2.) Fluorescence – ground state to single state and back.Phosphorescence - ground state to triplet state and back.

Spins pairedNo net magnetic field

Spins unpairednet magnetic field

10-5 to 10-8 s 10-4 to 10 s

Fluorescence Phosphorescence

0 sec 1 sec 640 sec

Example of Phosphorescence

3) Jablonski Energy Diagram

S2, S1 = Singlet StatesT1 = Triplet State

Resonance Radiation - reemission at same usually reemission at higher (lower energy)

Numerous vibrational energy levels for each electronic state

Forbidden transition: no direct excitation of triplet state because change in multiplicity –selection rules.

4.) Deactivation Processes:

a) vibrational relaxation: solvent collisions- emission > excitation (Stokes shift)- 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

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.

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.

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 (): 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

= kf

kf + ki + kec+ kic + kpd + kd

f: fluorescence I: intersystem crossingec: external conversion ic: internal conversionpd: predissociation d: dissociation

6.) Types of Transitions:- seldom occurs from absorbance less than 250 nm

‚ 200 nm => 600 kJ/mol, breaks many bonds

- fluorescence not seen with - typically * or * n

Increase quantum yield by decreasing factors that promote other processes

7.) Fluorescence & Structure:- usually aromatic compounds

‚ low energy of * transition ‚ quantum yield increases with number of rings

and degree of condensation. ‚ fluorescence especially favored for rigid

structuresfluorescence increase for chelating agent

bound to metal.

N HN

H2C

N

O

Zn

2

Examples of fluorescent compounds: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.

NH H

NH H

NH H

resonance forms of aniline

Fluorescence pH TitrationFluorescence pH Titration

9.) Effect of Dissolved O2:- increase [O2] decrease fluorescence

‚ oxidize compound ‚ paramagnetic property increase intersystem

crossing

B) Effect of Concentration on Fluorescence or Phosphorescence

power of fluorescence emission: (F) = K’Po(1 – 10 –bc) K’ ~ (quantum yield) Po: power of beam bc: Beer’s law

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

At low concentrations: F = 2.3K’bcPo

deviations at higher concentrations can be attributed to absorbance becominga significant factor and by self-quenching or self-absorption.

Fluorescence of crude oilFluorescence of crude oil

C) Fluorescence Spectra

Excitation Spectra – measure fluorescence or phosphorescence at a fixed wavelengthwhile varying the excitation wavelength.

Emission Spectra – measure fluorescence or phosphorescence over a range of wavelengths using a fixed excitation wavelength.

Phosphorescence bands are usually found at longer Phosphorescence bands are usually found at longer (>(>) then fluorescence because excited triple state is ) then fluorescence because excited triple state is lower energy then excited singlet state.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

Ion Reagent Absorption (nm) Fluorescence (nm) Sensitivity (g/ml) Interference

Al3+ Alizarin garnet R 470 500 0.007Be, Co, Cr, Cu, F-,NO3-, Ni, PO4

-3, Th, Zr

F- Al complex of Alizarin garnet R (quenching)

470 500 0.001

Be, Co, Cr, Cu, F-,Fe, Ni,PO4-3, Th, Zr

B4O72- Benzoin 370 450 0.04 Be, Sb

Cd2+ 2-(0-Hydroxyphenyl)-benzoxazole

365 Blue 2NH3

Li+ 8-Hydroxyquinoline 370 580 0.2 Mg

Sn4+ Flavanol 400 470 0.1 F-, PO43-, Zr

Zn2+ Benzoin - green 10B, Be, Sb, colored ions

N

OH

O

O

OH

OH

HO N N

HO

SO3Na

C

O

C

H

OH

8-Hydroxyquinoline flavanol alizarin garnet R benzoin

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 + hExamples:

C

NH

NH

C

NH2 O

O

O2/OH-

NH2

COO-

COO-

+ h + N2 + H2O

1) Chemical systems- Luminol (used to detect blood)

- phenyl oxalate ester (glow sticks)

2) Biochemical systems- Luciferase (Firefly enzyme)

Luciferin + O2

LuciferaseO C

O O

C R2

R1

SpontaneousCO2 + O C*

R2

R1

Light

S

N

HON

S

O

HO

Luciferin (firefly)

“Glowing” PlantsLuciferase gene cloned into plants