Electronic Spectroscopy Absorption and Emission

7/27/2019 Electronic Spectroscopy Absorption and Emission

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Electronic Spectroscopy: Absorption and emission

Interaction of e.m. waves with a material systems: selection rules.

Absorption spectroscopy

Emission spectroscopy in the frequency domain

Examples of molecules used in biology for emission experiments

Fluorescence quantum yield

Fluorescence quenching

Time resolved fluorescence

FRET: Fluorescence resonant energy transfer

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Matter - e.m. wave interaction(1)

Molecular orbitals and the corresponding electronic energy states:

Groundstate: Eg

a

bc

de

f Eg = 2Ea+ 2Eb+ 2Ec

Ee1 = 2Ea+ 2Eb+ Ec +Ed

Ee2 = 2Ea+ 2Eb+ Ec + Ef

Excitedstate: Ee1Excitedstate: Ee2

Energy of the electronic state and electronic transitions

Increasing

Energy

Eg

Ee1

Ee2

Transition Energies:

E = Ee1Eg=EdEc

E = Ee2Eg=EfEc

NB: The molecular orbital c and d are called HOMO(Highest Occupied Molecular Orbital) and LUMO(Lowest Unoccupied Molecular Orbital ), respectively.


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Matter - e.m. wave interaction(1)

Absorption spectroscopy correspond of an attenuation of an e.m. wave travelingin a material sample, because ther is transfer of energy from the e.m. wave tothe material.

This interaction occurs only if these conditions are satisfied::

1. Resonance condition: The energy of the photons of the e.m. wave must be

equal to the energy difference between two electronic levels of the materialsystem.

2. Selection Rule: Not all electronic transitions can give rise to absorption ofe.m. waves, because there are physical constraints that must be satisfied tohave transfer of energy between the e.m. wave and matter.

0...... EEhE imermer ==

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Stato

eccitato

Stato

fondamen

tale

s s

s* p

From a microscopic point of view, interaction with e.m. waves occurs only ifthere is a net change in the charge distribution between the two electronicstates involved in the transition so as to create a transition dipole on themolecule.

Creation of a dipolemoment in thetransition.

Eint= E e

The electric fieldinteracts with thecharges inside themolecule. The charge

distribution isapproximated with adipole:

Selection Rule: Matter - e.m. wave interaction(2)


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The electric field interacts with the charges inside the molecules. Weapproximate the charge distribution to a dipole. The inetraction energy

V(t) i given by:

)()( tEtVr

r

=

drrr ir

rfi = )()(

*

This expectation value is called transition dipole momentbetween the initial and final state and MUST BE DIFFERENTFROM ZERO:

Dipole moment associated tothe molecule

Electric field of the e.m. wave

The material system is characterized by quantized energy levels (electronic or vibrational)The dipole moment value will then be computed as an expection value integrated over allthe possible distribution of charge (electron and nuclei) density of the quantized energystates, and is therefore expressed as:

where (r) are the wavefunction that describe the initial and final electronic states.

Selection rule: Matter - e.m. wave interaction(3)

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h h

Influence of electronic spin : Selection Rule

One photon can be absorbed by the matter only if the inital state (g) andthe final state (e) have the equal total spin moment (the sum of all thespins of each elctron is qual)

=i

sTOT im )(

0=TOT 0=TOT0=TOT

1=TOT

singletTriplet

Sg SeTeSg

Selection rule: Matter - e.m. wave interaction(4)


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Interaction with an e.m. wave (green arrow) with resonant frequency, givesrise to absorption towards an excited state Se2, that means molecules arepromoted to the excited energy state Se2. This is a non-equilibrium state for themolecules, that tends to return to the ground state Sg by releasing theabsorbed energy thorugh different type of physical processes that can beraditive (stright colored arrows) or non radiative (wavy arrows) .

Emission Processes

Se2

Se1

Sg

Te1

The emission frequencies have discete values, and it can be noticed beacusethe emitted light is colored (only some frequencies) and not white (continouslight).

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The probability that the energy release for the excited molecules goes through aradiative process is ruled by the same selection rules that are valid forabsorption processes, i.e.:

1. The emission energy of the photons (h) must be equal to the trasnitionenergy between two electronic state of the system (resonance condition)

2. There must by generation of a dipole moment between the chargedistribution of the initial and final electronic states involved in the transition(not all possible electronic transition can give radiative emission)

3. Transition occurs only between electronic state with the same total spinmomentum

Condition 3 is often removed in real molecular systems, and we can observeemission from excited singlet and triplet states towards the ground state.

Emission Processes


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Abs./Em.

Se2

Se1

Important: For almost all molecular systems with rare exceptions, it is possible to

have absorption from Sg towards many excited states Se1, Se2 etc., whereasemission can come only from Se1. (Kasha Rule: radiative emission is more likelythe larger the energy distance between the two singlet electronic states). Fromall the higher excited state non radiative processes are dominant

It follows that the emission spectrum does not depend on the excitation

wavengeth choosen.

Se2

Se1

Sg

Emission Properties

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Example:

Emission spectrum ofa chlorophyll

Emission Properties


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Spectral shape of the emission spectrum

E

r

The absorption of e.m. wave promotes themolecule in vibrational excited stated of theelectronic state. The decay occurs throughtwo sequential processes :

1. Non radiative vibrational relaxation thevibrational energy is tranfered to thesurrounding as heat

2. Radiative emission (fluorescence) from thelowest vibrational level of Se1 towards allvibrational levels of Sg

Emission properties

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00

Sg

Se1

Se2

00

Sg (v = 0) S1

v = 0v = 1v = 2..

Absorption spectrum:Vibrational structure of Se1

S1 (v = 0) S0v = 0v = 1v = 2..

Fluorescence spectrum:Vibrational structure of Sg

Mirror Simmetry bewteen absorption and emission


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300 400 500

0,0

0,5

1,0

1,5

2,0

448

423

400379

375.4356.2

339

323.5

309.2

Intensita'(a.u.

)

lunghezza d' onda (nm)

Antracene in etanoloassorbimento

fluorescenza

The spectra of anthracene show clearly the vibrational contribution:Absorption spoectrumexcited state vibrational frequency = 1436 cm-1

Emission spectrum ground state vibrational frequency = 1385 cm-1

Example: Emission Properties

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Fluorimeter

Lamp

Excitation

Monochromator

Sample cell

Emission

Monochromator

Detector:

Photomultiplier Tube

Emission Properties


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exc

If

em

Fluorescence (emission) spectrum

Fluorescence Excitation Spectrum

Excitation at fixed ecc.Fluorescence intensity measured as afunction of the emission wavelength

Emission at fixed em. scansioneFluorescence intensity measured as a function

of the excitation wavelnegth. Gives the sameresults as absorption spectra, usefull foropaque or turbid samples.

If

Emission Properties

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Non radiative decay: Internal Conversion

The excitation energy is lost through a decay process that involves only thevibrational states of the molecules. There is transfer of energy form thevibrational states of the excited electronic state to the vibrational states of theground electronic state.

To have CI it is necessary an overalop between the vibrational wavefunctions in thetwo different excited electronic states.

Se

1

S0

R

E IVR

IC

Sg

IVR = internal vibrationalrelaxation: vibrational decayinside an electronic state.

IC = inetral conversion:Vibrational decay involving in twodifefrent electronic states

In both cases energy isdissipated as heat inthe systemenvironment.

Non radiative decay


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The selection rule that states that radiative decay can occur only betweenelectronic states with equal total spin momentum is not a strict one and can beovercome if there is mixing betwween the electronci states with different total spinmomentum. For this reason radiative emission occurs also from triplet excitedstates:

Se1

Te1

Sg

ISC: inter System Crossing

exc

fluorescence phosphorescence

Emission Process

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Phosphorescence arises from:

1. Excitation of the molecule in a excited singlet electronic state2. Internal vibrational relaxation in the excited singlet electronic state3. ISC: Inter System Crossing energy transfer from th excited singlet to the

excited triplet electronic state4. Radiative decay from the excited triplet electronic state to the ground singlet

electronci state.

Sonce thee excitedtriplet state falls at

lower energies thatthe excited singletelectronci state, in theemission spectrumphosphorescenceappears at higherwavelengths (shorterfrequencies) withrespect tofluorescence.

(nm)

Intensity(a.u.)

Absorption Fluorescence

Phosphorescence

Emission Processes


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Organic molecules without heayatom do not displeyphorsphorescence at roomtemperature, because the non-radiative decay form the tripletexcited state is the dominantprocess.

To observe phosphorescence it isnecessary to lower the temperature(77K liquid nitrogen) becausethen IC is reduced; or add heavyatoms to the molecule (for example

Br and I or transition metals)beacuse the latter enhance themixing between singlet and tripletelectronic states.

Phosphorescence

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Emission Processes

FLUORESCENCE QUANTUM YIELD (FQY) quantifies the intensity of thefluorescence or better the efficiency of the radiative decay processes with respectto all other non-raditive decay processes and is defined as:

ph

abosrbed

ph

emittedFQY

N

N =

Setting kR and kNR as the probability per unit time that a molecule in an excitedelectronic state will decay to the ground electronic state through a raditive or non-raditaive process, respectively , then the FQY can be expressed as:

NRR

RFQY

kk

k

+=

NB kR and kNR have unit of measurments equal to s-1. the maximum value

for the FQY is 1.

FQYfluI

FQY is strongly dependent on the physicsal parameters (temeprature) andchemical nature of the environemnt (solvent, pH, impurities etc.) that surroundthe molecule.

photonsofnumeroNph=


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Fluorescence Quenching

The probability for radiative emission can be changed by interaction with externalagents that add a new decay route for the excited molecule, thereforedecreasing the fluorescdnce quantum yield.

These external agents can be other molecules that can absorb the energy of theexcited molecules or change its chemical nature (charge transfer processes).

They are generically called quencher Q

QQ

QSQS

SS

exc

exc

ge

e

h

g

++

1

1

Se1

Sg

Qexc

Q

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Fluorescence quenching occurs as acollision bewteen the excitedmolecule and the quencher, thecollision i istantameous and allowsinteraction bewteen the two units.

As a consequence the probability tohave quenching is equal to theprobability to have a collision and the

latter depends on how fast thequenche molecules move and on their

concentration [Q].

Dynamic quenching

[ ]

[ ][ ]Q

kk

k1

kk

Qkkk

Qkkk

k

kk

k

NRR

Q

NRR

QNRR

Q

FQY

FQY

QNRR

RQ

FQY

NRR

RFQY

++=

+

++==

++=

+=

Q

flu

flu

I

I

Q

FQYFQY /

The ratio of the FQY withoutQ and the FQY with Q is linearin [Q]

Fluorescence Quenching


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Fluorescent systems

Triptophane (Trp) exposed to water emits at 350 nm,wehereas Trp inside the hydrophobic core of the protein emitsat 330 nm with dtronger inetnsity

Tyrosine (Tyr) gives a relevant contribution to fluorescencebecause it is present in large amounts.

Tyr fluorescence can be quenched by Trp through energytransfer

Phenylalanine (Phe) fluorescence an be observed only ifthere is no Trp and Tyr in the surrounding of Phe

Intrinsic Fluorescence of proteins

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0.04282 nm200257 nm6.4 nsPhenylalanine

0.14303 nm1400274 nm3.6 nsTyrosine

0.20348 nm5600280 nm2.6 nsTryptophan

QuantumWavelengthAbsorptivityWavelength

FluorescenceAbsorptionLifetimeAmino acid

Fluorescent systems


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ANS (1-Anilino-8-napthalene sulphonate

(proteine)

Fluorescin (proteine)

Ethidium bromide(DNA)

Acridine orange(DNA)

A fluorescent probe is a fluorescent molecule desigend with specific groups thatbind it to a well defined biological macromolecule or that can recognize

specific agents.

Extrinsic Fluorophores Fluorescent systems

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O O-O

O

O2Na+

Fluorescein

N O O

CF3

Coumarin 540A

Rhodamine B

O N+(CH2CH3)2(CH3CH2)2N

COOH

Cl-

Fluorescent molcules can be recognizedbecause their emission falls at differentwavelnegths.

Extrinsic Fluorophores Fluorescent systems


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Utikity of fluorescence quenching

Trp is quenched by iodide present in thewater solution. Only Trp units exposed towater and not inserted inside idrophobiccavities can therefore by quenched.

Fluorescent systems

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Examples of quenchersused to quench thefluorescence of welldefined fluorophores.

Fluorescent systems


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Molecular Oxygen is a very efficientfluorescence quencher for allmolecular systems.

2-methylanthracene is inserted in lipidmembranes: DMPC e DPPC.

The investigation of the quenchingkinetic of 2-methylanthracene bymolecular oxygen gives information onthe diffusion speed of O2 inside theselipid membranes.

Fluorescent systems

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Green Fluorescing Protein

GFP is a protein extracted from the jelly fish Aequorea Victoria.

The fluorescence properties of this protein arise from the presence of afluorophore inserted inside the proteic structure, that is therefore wellshielded by external perturbations that can cause fluorescence quenching.

Fluorescent systems


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The fluorophore inside the protein is generated by a spontaneous structuralrearrangement of the protein itself, without need of enzymatic stimuli.

The extended double bond structure and the presence of electron-withdrawingand electron-donating groups inside the fluorophores move the absorption andthe emission of this molecular unit in the visible.

Fluorescent systems

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It is possible to synthesize mutants ofGFP fluorescing in different spectralregions: RFP (red fluorescent protein) andYFP (yellow fluorescent protein).Because of the high bio-compatibility ofthese systems, they are largely used inbio-medical imaging.

Fluorescent systems

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Electronic Spectroscopy Absorption and Emission