MOLECULAR FLUORESCENCE SPECTROSCOPY - … and phosphorescence which are produced by different mechanisms. •Fluorescence is distinguished from phosphorescence by the lifetime of the

MOLECULAR FLUORESCENCE SPECTROSCOPY

Prepared by:

Dr.Elsadig H.KH.Adam

Instrumental Analysis -Dr.Elsadig H.k.Adam1

• Fluorescence is a form of photoluminescence; and thislater is a type of luminescence that occurs when certainmolecules are excited by electromagnetic radiation andas a consequence remission of radiation either of thesame wavelength or longer one takes place.

• The two most common photoluminescence arefluorescence and phosphorescence which are producedby different mechanisms.

• Fluorescence is distinguished from phosphorescence bythe lifetime of the excited state, with fluorescence theexcited state ceases immediately after irradiation isdiscontinued, (10-7 s), while phosphorescence continuedfor a detectable time (100 s).


Theory of molecular fluorescence

• An excited molecule can return to its groundstate by combination of several mechanisticsteps. Deactivation or relaxation processes canbe classified to radiative and nonradiativeprocesses.



Radiationless deactivation;

1-Vibrational relaxation (VR)

Conversion of the excited electron from the highest energy sublevel to the lowest energy sublevel in the same main energy level.

2-Internal conversion (IC):

It is intermolecular processes by which a molecular passes from anelectronic excited energy level (S2) to another lower excited energylevel (S1).

3-External conversion (EC):

It is deactivation of an excited electronic state which involveinteraction and energy transfer between the excited molecules and thesolvent or other solutes.Instrumental Analysis -Dr.Elsadig H.k.Adam5

• Intersystem crossing (ISC) is a process in whichthe spin of an excited electron is reversed. Theprobability of this transition is enhanced if thelowest vibrational energy level of the lowestexcited singlet state is almost identical in itsenergy to that of the triplet excited state.

• ISC is common in molecules containing heavyatoms such as iodine and bromine, also it isenhanced in presence of paramagnetic moleculessuch as molecular oxygen.


Radiative deactivation:

1-Fluorescence

Transition from S2 or S1 to the ground singlet state (S0)occurs with loss of energy in the form of EMR (emissionof photons) is termed fluorescence (S1 or S2-S0).

2-Phosphorescence

Phosphorescence occurs when an electron in anexcited triplet state relaxes to the ground singlet statewhile emitting radiation (T1 – S0).


Excitation and emission spectra:

If the intensity of emitted light (fluorescence) at afixed wavelength (emission) is plotted as a functionof wavelength of radiation used to excite amolecule, an excitation spectrum will result.

On the other hand, if the intensity of emittedradiation (fluorescence) is plotted versuswavelength, an emission spectrum is obtained. Inthis case, the sample is irradiated withmonochromatic radiation of certain wavelength(excitation) and a scan of the wavelength of emittedradiation is recorded.


If both of the excitation and emission spectra of a compound areplotted on the same chart, the following will be observed: 1-displacement of emission band to longer wavelength (Stock’sshift). 2- excitation and emission spectra bear a mirror imagerelationship to each other as shown in the following figure.

Excitation and emission spectraInstrumental Analysis -Dr.Elsadig H.k.Adam9

Quantum yield ():

The quantum yield or quantum efficiency () for a fluorescentprocess is the ratio of the number of molecules that fluoresce tothe total number of excited molecules or the ratio of number ofphotons emitted to that absorbed. For a highly fluorescentmolecule approachs unity ( = 1), while for a nonfluorescentmolecule =0.

Quantitative fluorimetry:

F = 2.3 K bc I0

F = K/ c

A plot of fluorescence intensity versus concentration is linear atlow concentration. When the concentration becomes highenough, A > 0.05 linearity is lost.


At high concentration, two main factors are responsiblefor deviation from linearity:

1-Self-absorption: this occurs when the wavelength ofemission overlaps with an absorption peak. Then, someof the emitted radiation will be absorbed by molecules insolution and a decrease in fluorescence takes place.

2- Self-quenching: it results from the collision of theexcited molecules.


Factors affecting fluorescence:

1- Molecular structure• The most intense and most useful fluorescent behavior is

found in compounds containing aromatic functionalgroup. Compounds containing aliphatic and alicycliccarbonyl groups or conjugated double-bond structuresmay also exhibit fluorescence.

• The quantum yield increases with the increase of numberof fused rings. The simplest heterocyclics, such aspyridine, thiophene, pyrrole and furan do not fluoresce(the lowest transition is n - * system which is rapidlyconverted to triplet and prevents fluorescence).

• Halogen substitution especially with bromine and iodineresults in a decrease in fluorescence due to intersystemcrossing.


• Fluorescence is favored in molecules that posses rigid planarstructure. For example fluorene fluoresce much more intensethan biphenyl due to rigidity furnished by methylene group influorene. The influence of rigidity is accounted for theincrease of fluorescence of certain chelating agents whenthey form complexes with a metal ion e.g. the fluorescentintensity of 8-hydroxyquinoline is much increased when itforms zinc complex.

CH2

N

O

n2

Zn

Fluorene BiphenylThe zinc complex


2- Effect of temperature and solvent:• The quantum efficiency of fluorescence by most molecules

decreases with increasing temperature, as deactivation byexternal conversion is favored. Also a decrease in solventviscosity leads to the same result.

• Polar solvents may enhance fluorescence, while it isdecreased by solvents containing heavy atoms such as carbontetrabromide or ethyl iodide.

3- Effect of dissolved oxygen:

Being paramagnetic, dissolved oxygen decreases the

fluorescence due to intersystem crossing.


InstrumentationIt is composed of the following main parts shown inthe following diagram

Schematic diagram of a spectrofluorimeter


1-Source of energySeveral sources have been used, the two mostcommonly used are:A-Mercury–arc lamp:It is a quartz lamp containing mercury vapor whichupon electrical excitation emits line spectra ofseveral definite wavelengths. It can not be usedwhen a scan of spectrum is required.B-High pressure xenon lamp:This lamp emits a continuum of radiationthroughout the UV-Vis region so it is useful whenspectrum scanning is required.


2-Wavelength selectorTwo filters (either absorption or interference filters can beused) or monochromators (grating type) are used; onebetween the source and the sample and the other betweenthe sample and the detector.

3-The cell:Tetragonal or cylindrical transparent, glass or quartz tubesare used (the four sides are transparent).

Compare with the sample cell in the spectrophotometry.

4-Detectors and readout meter:

Photomultiplier type is used since the intensity of emittedradiation is small. Digital or analog or null point meter areused.


Important notes:• Emission of radiation by sample takes place in all

directions. The emitted radiation is measured at 900

from the path of the exciting beam and at the center ofthe cell.

• This is to minimize the error due to scattering of lightfrom the walls of the cell and to prevent theinterference from the exciting beam, which occurs atother angles.

• Since a broad emission band is obtained, it isnecessary to use a second wavelength selectorbetween the sample and the detector in order to passthe most intense emitted wavelength (emission).


Applications of fluorimetry:• Compounds which are intrinsically fluorescent are

easily determined at very low concentrations by simplefluorimetric method (Direct fluorimetry). For example,phenobarbitone, quinine, emetine, adrenaline,cinchonine, reserpine vitamin A, riboflavine and othernatural products.

• Nonfluorescent substances can be determined afterchemical reaction (Indirect fluorimetry).

• Inorganic ions can be determined either by formationof fluorescent chelates upon reaction with fluorimetricreagents e.g. 8- hydroxyquinoline (for Al3+), benzoin(for Zn2+) or flavanol (for Zr3+) or by measuring thequenching of fluorescence of a fluorescent substancein the presence of some ions.


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MOLECULAR FLUORESCENCE SPECTROSCOPY - … and phosphorescence which are produced by different mechanisms. •Fluorescence is distinguished from phosphorescence by the lifetime of the