Fluorescence Spectrometry
(SPECTROFLUOROMETRY)
is an the energy transition from
Fluorescence emission phenomenon,
a higher to lower state within the molecule concerned being
measured by the detection of this emitted radiation rather than the
absorption.
A molecule absorbs light at one wavelength and reemits light ata
longer wavelength.
An atom or molecule that fluoresces is termed a fluorophore
Fluorometry is defied as the measurement of emitted florescent
light.
I. Principles of Fluorescence
1. Luminescence
Emission of photons from electronically excited states
Two types of luminescence:
[Link]
excited state
Relaxation from singlet
2. Phosphorescence
Relaxation from triplet excited state
I. Principles of Fluorescence
2. Singlet and triplet states
Ground state two electrons per orbital; electrons have
and are paired
opposite spin
Singlet excited state
Electron in higher energy orbital has the
relative to electron in the lower orbital
opposite spin orientation
Triplet excited state
The excited valence
electron may spontaneously reverse its
flip). This process is called intersystem crossing. Electrons in spin
both
(spin
orbitals now have same spin orientation
I. Principles of Fluorescence
3. Types of emission
Fluorescence return from excited singlet state to
groundstate does not require change in spin
orientation (more common of relaxation)
Phosphoresence return from a triplet excited state to
a ground state; electron requires change in spin
orientation
Emissive rates of fluorescence are several orders of
magnitude taster than that of phosphorescence
I. Principles of Fluorescence
(10S) (10"S) (10S-10S)
Total spin S 0
IC (AS-0) Multiplicity M
1
IsC 1AS=0) (10-10?S)
IC (AS-0)
hVp
So
Absorbtion Fluorescence Phosphorescence
0) (AS- (AS 0 )
Jablonski Diagram
Basic concepts
Each molecule contains a series of closely spaced energy levels.
Absorption of quantum of light energy by molecule causes the
a a
transition of an electron from the singlet ground state to one of a
number of possible vibrational levels of its fist singlet state.
The actual number of molecules in the excited state under typical
reaction conditions and excited with a typical 150-wW light source is
very small and is estimated to be about 10 -13 mole per mole of
fluorophore.
Basic concepts
is in an excited state, it returns to its
Once the molecule
original energy state in several ways.
(1) radiation-less vibrational equilibration(1C)
state
(2) the florescence process from the excited singlet
(3) quenching of the excited singlet state,
(4) radiationless crossover to a triplet state(ISC),
(5) quenching of the fist triplet state
(6) the phosphorescence process of light
emission from the triplet state
Internal conversion Vs intersystem crossing
Internal conversion is a transition
ocCurring between states of the same
it takes time
Iterystem
multiplicity and place at a
scale of 1012 s (faster than that of CrOsIng
fluorescence process)
Intersystem crossing
-
Intersystem crossingbetween
refers tostates
non- of
radiative transition
different multiplicity hsorption
It IUOTesCeke
occurs via inversion of the spin of the
excited electron resulting in two unpaired parsptorexcence
electrons with the same spin orientation,
resulting in a
state with Spin=l and
multiplicity of 3 (triplet state)
Heat
Stokesshift
Excltation Emission
IA Energy levels
Wevelength of lghe (nm)
The difference between the maximum wavelength of the excitation light and
maximum wavelength of the emitted florescence is constant
light a
the
referred to as the Stokes shift.
Measure of energy lost during the lifetime ofthe excited state (radiation-
less vibrational deactivation) before returning to the ground singlet level
(forescence emission
The best results are obtained from compounds involving large shits.
sintsS.
P "VO g d9e
Relationship of Concentration and
Fluorescence Intensity
h e relationship of concentration to the intensity of fiorescence emission is derived from
the Beer-Lambert law
By expansion through a laylor series, rearrangement, logarithm base conversion, and basic
dssumpton's dDout chute olutions, the tollowing equation is obtained:
w here
F=oll(2.3-abc)]
F-te at ve fiorescence ntesty
O- tiorescence etthency (ie. the ratio between quanta of ight em tted and quanta of
lght absorbed)
i aexcitatior intensity
V O u m e element cered Dy ceomery or tne extitation and eI5SIon sits
e in
cOmceniranO moly
FHorescence intensity is directly proportional to the concentration of the tluorophore and the
exCitdtion intensity.
This relationship holds only for dilute solutions, in which absorbance is less than 2% of the
Exciung raciation
Auerescenee lintensly
cencentratiens
A t low concentrations of fluorophore, the fluorescence
sample is essentially linearly proportional to concentration.
intensity of a
However, as the concentration increases, a point is reached at which
the intensity
increase is progressively
less linear, and the intensity
eventualy decreases as concentration increases further.
Endogenous Excitation Emission
maxima (nm) maxima (nm)
Biological Fluorophores Amino acs
Tryptophan 275 O0
yrosine
Pheny
ra protcins
325 400, 405
Endogenous Fluorophores Elastin 90, 525 340, 400
y m e s and coenzymes
amino acids
AD, lavins
NAD
structural proteins NADPH
itamins
336
enzymes and co-enzymes Vitamin K
Vitamin D 390 80
vitamins itamin Ba compounds
Pyndoxine
lipids Pyridoxamine
Pyridoxic acid 315
porphyrins Pyidoxal3 phosphate
Vitamin Bi2
330
275
00
05
Exogenous Fluorophores Lipids
hopholprds
Cyanine dyes Ceroid 40--395 430-460. 540
Porphyrins AN=
45 630, 60
Photosensitizers
AD
denine dinucleotide: ANDIPLL redoed ticoina
Molecular markers GFP, etc. nuckeotide phosphate.
IV. Biological Fluorophores
ALEMeard fiereace lfetune and amplnudes for endogrnoun buological fuorophores in vtro
3971 Hi KCitatuonl, wath coupariE to etanae v a j
hphore
X10 M In
Meaua Literatue
B Latete s ) Area ifetane i a AIA
RetereKes
Tnptopha
Elmtan 16 7 36 6
4 67 0 8 042
657 027 073
FAD 0
*
NADH hee 6
s 077 023
4
097 003
0 3 8
Rel. JD Pits ard M A Mycek Hevlew d Soernic inirumernts.
Vol 72. No7, pp 3061-3072 (2001) UM BiomedE B00 Bomedical Ogtics Fal 2003 PrG Myces
