fluorometry manik

Luminescence: Luminescence is the phenomenon of a

chemical species to absorb radiation of UV or visible

region and emit a radiation of longer wavelength. Loss of

energy and concomitant transition of molecules from

excited states to ground states with emission of radiation is

called luminescence.

Luminescence can be divided into two types depending

on the lifespan of the excited state –

1. Fluorescence

2. Phosphorescence

Singlet and triplet states

• Ground state – two electrons per orbital; electrons have opposite spin and are paired

Singlet excited state

Electron in higher energy orbital has the opposite spin orientation relative to electron in the lower orbital

Triplet excited state

The excited valence electron may spontaneously reverse its spin (spin flip). This process is called intersystem crossing. Electrons in both orbitals now have same spin orientation.

Types of emissionFluorescence – return from excited singlet state to ground state; does not require change in

spin orientation (more common of relaxation)

An atom or molecule that fluoresces is termed a Fluorophore

Fluorometry is defined as the measurement of the emitted fluorescence light

Phosphoresence – return from a triplet excited state to a ground state; electron requires change

in spin orientation

Vibrational energy level: Whether the molecule is in ground state orexcited state, the molecule contains many energy levels which are calledvibrational energy levels.

Vibrational relaxation: Vibrational relaxation is the transition of moleculefrom any of the vibrational energy levels to the lowest vibrational energylevel of the excitatory state.

Resonance fluorescence: Resonance fluorescence is the phenomenonwhere the molecule absorbs and emits equal amount of energy. Practicallyresonance fluorescence doesn’t occur or occur rarely as vibrationalrelaxation occurs.

Internal conversion: The phenomenon of excited molecule to return to theground state by losing energy by means other than photo radiation istermed internal conversion.

Intersystem crossing: The transfer of a molecule present in the lowestvibrational energy level of the excited singlet state to an excited triplet stateis called intersystem crossing.

Property Fluorescence PhosphorescenceTransition Molecule transits from excited singlet state

to ground state.Molecule transits from excited tripletstate to ground state.

Lifespan Fluorescence is continued for only 10-8 to 10-4 seconds.

Phosphorescence continues for 10-4

seconds to 10 seconds.Afterglow Not present. Occurs and luminescence slowly fades.

Analytical application Yes. No.

Quantum efficiency: Quantum efficiency is defined as the ratio of number of light quanta emitted and the

number of light quanta absorbed.

Its significance is that, it is an indicator of how fluorescent a molecule is. If Q is near 1, the molecule is highly

fluorescent molecule and if Q is near 0, the molecule is a very low fluorescent molecule.

absorbedlight ofEnergy

emittedlight ofEnergy

absorbed quantalight of No.

emitted quantalight of No.orQ

The method of analysing a sample by measuring its fluorescence i.e.intensity and composition of light emitted by it, is called fluorometry.

Fluorescence spectroscopy aka fluorometry or spectrofluorometry is ananalytical technique for identifying and characterizing minute amounts of afluorescent substance by excitation of the substance with a beam ofultraviolet light and detection & measurement of the characteristicwavelength of the fluorescent light emitted.

It is a spectrochemical method.

When energy is applied to certain molecules in the form of UV or visible

electromagnetic radiation, the molecules temporally transit to an

excited singlet state where the excited electron is in paired condition

with the ground electron. In the excited state, the molecules lose energy

in radiationless manner to descend to the lowest vibrational energy

level of the excited state. The excited state lasts only 10-8 to 10-4 seconds

and then the excited molecule will return to ground state by losing

energy through emitting radiation. This is termed fluorescence and the

emitted radiation is of longer wavelength.

By measuring the emitted wavelength we can determine the presence

and amount of a compound in a sample.

Relationship between fluorescence and chemical structure

Definite correlations between chemical structure and fluorescence can’t be made.

Degree of conjugation

Delocalization of electron

Electron donating groups

Exception

Relationship between fluorescence and

chemical structure• Electron withdrawing

groups

• Exception

Relationship between fluorescence and chemical structureMolecular geometry

Rigidity and planarity

cis-trans isomerism

Heterocylic compounds

Ionization

Complexation

ISS PC1 (ISS Inc., Champaign, IL, USA)

Fluorolog-3 (Jobin Yvon Inc, Edison, NJ, USA )

QuantaMaster (OBB Sales, London, Ontario N6E 2S8)

Concentration of fluorescing species

Presence of other solutes/impurities

Chemical quenching

pH of the sample solution

Stability of the sample compound (Degradation of Sample)

Solvent effect

Temperature

Features Absorption spectroscopy Fluoroscence spectroscopy

Theoretical considerationMeasurement of amount of light

absorbed.Measurement of intensity of fluorescence.

Wavelength of light used Which gives maximum absorption. Which gives maximum fluorescence.

Instruments Determines only the absorption of light.Determines absorption of light as well as

emission of radiation.

Light source Tungsten, H2-discharge lamp. Mercury arc lamp, Xenon arc lamp.

Cell used Silica cell. Glass and metal cells.

DetectorPhototube or photo multiplier is used to

detect the radiation absorbed

Emission filter is used to separate the

emitted light from the transmitted light.

ConcentrationConcentration depends on the molar

absorptivity.

Concentration depends on the

characteristics of the instrument.

Electrical transitionApplicable for both ππ* & nπ*

transition.

Not applicable for the compound

containing nπ* transition.

Experimental variables

temperature & Extraneous

solution

Not so restricted. Highly restricted.

Sensitivity and selectivity Less sensitive and less specific. More sensitive and highly specific.

Application in chemistry

Determination of metal ions

Separation and identification

Application in biopharmaceutics

Pharmaceutical applications

SensitivitySpecificityWide Concentration RangeSimplicity and SpeedLow Cost

Limitations of Fluorometry

Excitation spectrum and emission spectrum

The excitation spectrum is a measure of the ability of the impinging radiation to raise a

molecule to various excited states at different wavelengths. An excitation spectrum is recording

of fluorescence versus the wavelength of the exciting or incident radiation and it is obtained by

setting the emission monochromator to a wavelength where fluorescence occurs and scanning the

excitation monochromator. An excitation spectrum looks very much like an absorption spectrum,

because the greater the absorbance at the excitation wavelength, the more molecules are promoted to

the excited state and the more emission will be observed.

The emission (fluorescence) spectrum is a measure of the relative intensity of radiation given

off at various wavelength as the molecule returns from the excited states to the ground state.

The emission spectrum is recording of fluorescence versus the wavelength of the fluorescence

radiation, and it is obtained by setting the excitation monochromator to a wavelength that the sample

absorbs and scanning the emission monochromator.

Since some of the absorbed energy is usually lost as heat, the emission spectrum occurs at longer

wavelengths (lower energy) than does the corresponding excitation spectrum. If an emission spectrum

occurs at shorter wavelengths than the excitation spectrum, the presence of a second fluorescing

species is confirmed.

The absorption and emission spectra will have an approximate mirror image relationship if the

spacings between vibrational levels are roughly equal and if the transition probabilities are similar.

Energy level diagram showing why structure is seen in the absorption and emission spectra, and why the spectra seem

roughly mirror images of each other.

Mirror image rule

• Vibrational levels in the excited states and ground states are similar

• An absorption spectrum reflects the vibrational levels of the electronically excited state

• An emission spectrum reflects the vibrational levels of the electronic ground state

• Fluorescence emission spectrum is mirror image of absorption spectrum

S0

S1

v=0

v=1

v=2

v=3v=4v=5

v’=0

v’=1v’=2v’=3v’=4v’=5

References• Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York.

• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York. 3 Id., p. 7.

• Dr. Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular Biology, School of Medicine.

• G. K. Turner, "Measurement of Light From Chemical or Biochemical Reactions," in Bioluminescence and Chemiluminescence: Instruments and

Applications, Vol. I, K. Van Dyke, Ed. (CRC Press, Boca Raton, FL, 1985), pp. 45-47.

• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 51-57.

• Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, chap. 2.


• Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, pp. 23-26.


• Stotlar, S. C. 1997. The Photonics Design and Applications Handbook, 43rd Edition, Laurin Publishing Co., Inc., Pittsfield, MA, p. 119.

• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 63.




• Iain Johnson, Product Manager, and Ian Clements, Technical Assistant Specialist (May 1998 communication from Molecular Probes, Eugene,

Oregon).

• Fluorometric Facts: A Practical Guide to Flow Measurement, Turner Designs (1990), pp. 14-15.


• Fluorometric Facts: A Practical Guide to Flow Measurement, Turner Designs (1990), p. 21.

• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York., p. 28.

• Teitz Textbook of Clinical Chemistry and Molecular diagnosis (5th Edition)

• Dr.B.K.Sharma, Instrumental methods of chemical analysis.

• Gurdeep R Chatwal, Instrumental methods of chemical analysis

• http://en.wikipedia.org/wiki/Fluorescence

• http://images.google.co.in/imghp?oe=UTF-8&hl=en&tab=wi&q=fluorescence

• http://www.bertholdtech.com/ww/en pub/bioanalytik/biomethods/fluor.cfm

http://www.bertholdtech.com/ww/en pub/bioanalytik/biomethods/fluor.cfm

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