Fluorescence, Phosphorescence, & Chemiluminescence

A) Introduction
1.)

Theory of Fluorescence and Phosphorescence:

10-5 to 10-8 s fluorescence

10-4 to 10s phosphorescence

10-14 to 10-15 s
10-8 10-9s

M* M + heat

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

Fluorescence, Phosphorescence, & Chemiluminescence

A) Introduction
1.)

Theory of Fluorescence and Phosphorescence:

Method

Mass detection
limit (moles)

Concentration Advantages
detection limit
(molar)

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

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 paired
No net magnetic field

Fluorescence

Spins unpaired
net magnetic field

Phosphorescence

Example of
Phosphorescence
0 sec

1 sec

640 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

usually reemission at higher (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 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 (): 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 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
- typically * or * n

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 structures
fluorescence increase for chelating agent
bound to metal.

Examples of fluorescent compounds:

H
N

H2
C
O
Zn

N
2

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.

Fluorescence pH Titration

H
N

H
N

resonance forms of aniline

H
N

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: C781C787, 2006.

B) Effect of Concentration on Fluorescence or Phosphorescence

power of fluorescence emission: (F)

= KPo(1 10 bc)
K ~ (quantum yield)

Po: power of beam

bc: Beers law

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

Fluorescence of crude oil

At low concentrations: F = 2.3KbcPo

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

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

(> ) 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
Ion
Al

3+

Reagent

Absorption (nm)

Alizarin garnet R

Fluorescence (nm)

470

Sensitivity (g/ml)

Interference

0.007

Be, Co, Cr, Cu,

F-,NO3-, Ni, PO4-3,
Th, Zr

500

Al complex of Alizarin
garnet R (quenching)

470

500

0.001

Be, Co, Cr, Cu,

F-,Fe, Ni,PO4-3,
Th, Zr

Benzoin

370

450

0.04

Be, Sb

2-(0-Hydroxyphenyl)benzoxazole

365

Blue

NH3

8-Hydroxyquinoline

370

580

0.2

Mg

Sn4+

Flavanol

400

470

0.1

F-, PO43-, Zr

Zn2+

Benzoin

green

10

B, Be, Sb,
colored ions

F-

B4O72Cd2+
Li+

OH
N

OH

O
HO

HO
N

SO3Na

flavanol

OH

OH

8-Hydroxyquinoline

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 + h
Examples:
1) Chemical systems
- Luminol (used to detect blood)
NH2

NH2

COONH

O2/OH-

+ h + N2 + H2O

NH
C

COO-

- phenyl oxalate ester (glow sticks)

2) Biochemical systems
- Luciferase (Firefly enzyme)

Luciferin + O2

Luciferase
O

C
1

R2

Spontaneous

R2

CO2 +

Light

C*
R1

Glowing Plants

N
S

HO

Luciferase gene cloned into plants

S
N
O

Luciferin (firefly)
HO