FLUORESENCE SPECTROSCOPY

Dr Soniya Kasliwal
PRINCIPLES

 Fluorescence is an emission phenomenon where an energy

transition from a higher to a lower state is accompanied by
radiation. Only molecules in their excited forms are able to emit
fluorescence; thus, they have to be brought into a state of higher
energy prior to the emission phenomenon.
 The phenomenon whereby a molecule , after absorbing radiations
emits radiation of a longer wavelength is known as fluorescence.
This shift toward a longer wavelength is known as Stoke’s shift.
 Fluoresecence is an extremely short lived phenomenon of about
10-7 seconds
 The absorption of a photon occurs very quickly (10–15 sec) from the
lowest vibrational level of the ground state (S0) to higher
electronic and vibrational states (e.g., S1, S2). The electron
relaxes quickly from higher energy states (10–10 to 10–12 sec) to the
lowest vibrational level of the first excited state (S1). This is
known as internal conversion.
 In the absence of photochemical reactions, there are two general
paths for loss of excited state energy: radiative and nonradiative.
 Loss of energy in a radiative pathway involves release of a
photon. When the photon comes from the first excited singlet
state to the ground state, the light released is fluorescence. An
electron can also undergo intersystem crossing, that is, move to
an excited triplet state from the excited singlet state . The return
of the electron from this triplet state to the ground state may be
accompanied by release of a photon. This emission is referred to
as phosphorescence
STRUCTURAL FEATURES
 Aromatic molecules(Resonance)
 Fluoresecence increases with number of electrons
 Polycyclic aromatic compounds more fluoresecent
 Electron withdrawing substituents decrease fluoresecence (- Cl, -
I,-Br,-NO2 , -NHCOCH3, -COOH etc .
 Substituents which delocalize the electron (-NH,-OH,-F, NHCH3
) enhance fluorescence.
FLUOROMETRY :THEORY AND INSTRUMENTATION

 Fluorometry is an important analytical tool for the determination

of extremely small concentrations of substances which exhibit
fluorescence.Beer –Lambert law is also applied to fluorometry.

log I solvent = f Cb
I sample

f = absorptivity of the fluorescent material

C = Concentration of the substance
b = path length

F = k (I solvent - I sample )
log Fn = f Cb
Fn – F
INSTRUMENTATION
 a continuous source of radiant energy (mercury lamp or xenon
arc)
 A monochromator usually a prism P 1 to choose the wavelength
with which the sample is to be irradiated.
 A second monochromator P2 which placed after the sample
enables the determination of the fluorescent spectrum of the
sample.
 A detector usually a photomultiplier suited for wavelengths
greater than 500 nm
 An amplifier
APPLICATIONS
 There are many and highly varied applications for fluorescence
despite the fact that relatively few compounds exhibit the
phenomenon.
 The effects of pH, solvent composition and the polarisation of
fluorescence may all contribute to structural elucidation.
Nonfluorescent compounds are often labelled with fluorescent
probes to enable monitoring of molecular events. This is termed
extrinsic fluorescence as distinct from intrinsic fluorescence
where the native compound exhibits the property.
 Some fluorescent dyes are sensitive to the presence of metal ions
and can thus be used to track changes of these ions in in vitro
samples, as well as whole cells.
 Qualitative analysis
 Quantitative analysis (applications include assay of riboflavin,
thiamine hormones such as cortisol, oestrogen, serotonin and
dopamine, organophosphorus pesticides, tobacco smoke
carcinogens, drugs such as lysergic acid and barbiturates
,porphyrins cholestrol ,and even some metal ions)
 Studies on protein structure (FAD containing proteins)
 Intracellular free calcium concentration assay
Quin -2 ( an EGTA derivative ) and Fura-2 are there fluorescent
probes which allow us to assay intracellular free calcium
concentration. These probes are permeable to the plasma
membrane and upon entering the cytosol combine with calcium.

 Fluorescent probes and studies on membrane structure

Fluorescent probes such as anilonapthalene 8 sulphonate ( and N
–methyl 2 anilino -6 napthalene sulphonate contain both charged
and hydrophobic areas and therefore locate at the water lipid
interface.
 Assay of membrane potential
changes in membrane potential can be studied by using
fluorescent probes such as merocyanine -540.
 Fluorescent microscopy
spectrofluorometer when combined with a microscope allows the
determination of subcellular location of fluorescent compounds or
of materials which bind fluorescent dyes
 Kinetics and thermodynamics of the incorporation of a
particular subunit or substrate into a macromolecular assembly.
FLUORESCENCE SPECTRA AND STUDY OF PROTEIN STRUCTURE

 The fluorescence of a protein is solely due to the amino acids

Tryptophan,tyrosine and phenylalanine.
 Usually it is tryptophan which is studied most often.
 Decrease in polarity of the solvent pushes to shorter wavelength.
Extrinsic fluorescence
 Frequently, molecules of interest for biochemical studies are
nonfluorescent. In many of these cases, an external fluorophore
can be introduced into the system by chemical coupling or non-
covalent binding.
 Three criteria must be met by fluorophores in this context.
Firstly, it must not affect the mechanistic properties of the system
under investigation.
 Secondly, its fluorescence emission needs to be sensitive to
environmental conditions in order to enable monitoring of the
molecular events.
 And lastly, the fluorophore must be tightly bound at a unique
location
 Example : EtBr ,proflavin ,Acriflavin and Acridine orange.
 For protein studies ANS 1-dimethylaminopthalene
sulfonate(DNS) 2 –p- toluidylnapthalene-6 –sulfonate (TNS)
,fluorescein, rhodamine and dansyl chloride
Quenching
 Fluorescence quenching refers to any process that decreases the
fluorescence intensity of a sample.
 A variety of molecular association can result in quenching. These
include excitedstate reactions, molecular rearrangements, energy
transfer, groundstate complex formation, and collisional
quenching.
 A wide variety of substances act as quenchers of fluorescence.
Quenching by oxygen is due to its paramagnetic nature causes the
fluorophore to undergo intersystem crossing to the triplet state.