priniciples of fluorescence spectroscopy

7/31/2019 Priniciples of Fluorescence Spectroscopy

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CONTENTS

1. Introduction

2. Photophysics of Fluorescent Sensors

i) Photoinduced Electron Transfer

ii) Eximer Formationiii) Photoinduced Charge Transfer

iv) Fluorescence Resonance Energy Transfer

3. Factros Affecting of Fluorescent Intensity

i) Solvent

ii) Temperature

iii) Structural Rigidity

iv) ph

v) Concentration


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1.INTRODUCTION:Luminescence is the emission of light from

any substance occurs from electronically excited states.

Luminescence is divided into two categories-fluorescence andphosphorescence depending upon excited states

Fluorescence is a spectrochemically method

of analysis where the molecules of the analyte are excited by

irradiation at a certain wavelength and emit radiation of different

wavelength. The emission spectrum provides information for both

qualitative and quantitative analysis. When light of an appropriate

wavelength is absorbed by a molecule (i.e.,excitation),the

electronic state of the molecule changes from the ground state toone of many vibrational levels in one of the excited electronic

states. Molecules in these higher vibrational levels then relax to the

lowest vibrational level of the excited state. From the lowest

vibrational level, several processes can cause the molecule to relax

to its ground state. The most important path ways are

Intersystem Crossing: The energy of the singlet state overlaps

those of the triplet states, vibrational coupling can occur betweenthe two states. Molecules in the singlet excited state can cross over

the triplet excited state

Internal conversion: Direct vibrational coupling between theground and excited electronic states .This is a rapid process(10

-12

sec)

Fluorescence: Corresponds to the relaxation of the moleculefrom the singlet excited state to the singlet ground state with

emission of the light Fluorescence has short lifetime (~10

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sec) sothat in many molecules it can compete favorably with collisional

deactivation, intersystem crossing and phosphorescence. The

wavelength (and thus the energy) of the light emitted is dependent

on the energy gap between the ground state and the singlet excited

state.


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Phosphorescence: This is the relaxation of the molecule fromthe triplet excited state to the singlet ground state with emission of

light. Because this is a classically forbidden transition, the triplet

state has a long lifetime and the rate of phosphorescence is slow

(10-2

to 100 sec).

Collisional deactivation (external conversion) leading to nonradiative relaxation.

Figure: Electronic transition energy level diagram


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Photophysics of Fluorescent Sensors

i) Photoinduced Electron Transfer(PET):when a lone electron pair is located in an

orbital of the fluorophore itself or an adjacent molecule and the

energy of this orbital lies between those of the HOMO and LUMO,

efficient electron transfer of one electron of the pair to the hole in

the HOMO created by light absorption may occur, followed by

transfer of the initially excited electron to the lone pair orbital.

Such PET provides a mechanism for nonradiative deactivation of

the excited state leading to a decrease in emission intensity or

quenching of the fluorescence

Chelation-enhanced fluorescence(CHEF):Fluorescence lost as a result of PET

may be recovered if it is possible to involve the lone pair in a

bonding interaction. Thus, protonation or binding of a metal ion

effectively places the electron pair in an orbital of lower energy

and inhibits the electron-transfer process. The excited-state energy

may then again be lost by radiative emission. In the case of metal

ion binding, this effect is referred to as chelation-enhancedfluorescence (CHEF).

Figure: Mechanisms for PET (a) and CHEF (b) systems.


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ii) Excimer Formation:Where aromatic rings are

involved in weak interactions (such as -stacking) which bring

them within van der Waals contact distances, electronic

excitation of one ring can cause an enhanced interaction with itsneighbor, leading to what is termed an excited-state dimer or

excimer. .Excimer emission typically provides a broad

fluorescence band without vibrational structure.

iii)Photoinduced Charge Transfer(PCT):Electronic excitation necessarily

involves some degree of charge transfer, but in fluorophores

containing both electron withdrawing and electron donating

substituents, this charge transfer may occur over long distances andbe associated with major dipole moment changes, making the

process particularly sensitive to the microenvironment of the

fluorophore.

For example, cation complexation of an electron

donor group within a fluorophore, the electron-donating character

of the donor group will be reduced. The resulting reduction of

conjugation causes a blue shift of the absorption spectrum together

with a decrease of the molar absorptivity. In contrast, metal ionbinding to the acceptor group enhances its electron-withdrawing

character, and the absorption spectrum is thus red-shifted with an

increase of the molar absorptivity.


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Figure:Photoinduced Charge Trasfer System

iv)Fluorescence Resonance Energy Transfer:FRET arises from an interaction

between a pair of dissimilar fluorophores in which one acts as a

donor of excited-state energy to the other (acceptor). This returns

the donor to its electronic ground state, and emission may then

occur from the acceptor center.

FRET is influenced by three factors

1.The distance between the donor and the acceptor


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2.The extent of spectral overlap between the donor emission and

acceptor absorption spectrum

3.The relative orientation of the donor emission dipole moment

and acceptor absorption moment

Factros Affecting The Fluorescence Intensity:

priniciples of fluorescence spectroscopy

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