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Fluorescence Spectroscopy AK

Fluorescence spectroscopy is a sensitive optical emission technique that detects molecules by measuring the intensity of their emitted light after being excited by a photon source. Key factors affecting fluorescence include molecular structure, viscosity, temperature, and pH, while its applications span across inorganic and organic chemistry, including assays and environmental studies. Despite its advantages in sensitivity and specificity, limitations such as the need for careful sample handling and potential interference from contaminants must be considered.

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13 views19 pages

Fluorescence Spectroscopy AK

Fluorescence spectroscopy is a sensitive optical emission technique that detects molecules by measuring the intensity of their emitted light after being excited by a photon source. Key factors affecting fluorescence include molecular structure, viscosity, temperature, and pH, while its applications span across inorganic and organic chemistry, including assays and environmental studies. Despite its advantages in sensitivity and specificity, limitations such as the need for careful sample handling and potential interference from contaminants must be considered.

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Akshay Kumar -AK
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© © All Rights Reserved
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FLUORESCENCE SPECTROSCOPY

AND ITS APPLICATIONS

GUIDED BY:
SUBMITTED BY:

JAYARANI M AKSHAY KUMAR K


INTRODUCTION
 Fluorescence spectroscopy is a sensitive optical emission
technique in which sample molecules are excited with a photon
source. Those molecules that relax by radiant emission can be
subsequently detected by measuring the intensity of that emission.

 Fluorophores play the central role in fluorescence spectroscopy.

 Fluorophores are the components in molecules that cause them


to fluorescence.
PRINCIPLE OF FLUORESCENCE SPECTROSCOPY
• Absorption of UV or visible radiation causes transition of electrons from
singlet ground state to the singlet excited state.

• As this state is not stable, it emits energy in the form of UV or visible radiation
and returns to singlet ground state.

• Fluorescence emission occurs as the fluorophore decay from the singlet


electronic excited states to an allowable vibrational level in the electronic
ground state.

• The fluorescence excitation and emission spectra reflect the vibrational level
structures in the ground and the excited electronic states respectively.
• It is a diagram that illustrates the electronic states of
a molecule and the transitions between them.
• SPECTROFLOUROMETER mainly consists of ;

• Source of light
1. Mercury vapour lamp
2. Xenon arc lamp
3. Tungsten film
• Filters and monochromators
1. Primary filters and secondary filters
2. Excitation monochromators and Emission
monochromators
• Sample cells, Detectors
FACTORS AFFECTING FLUORESCENCE

1. Conjugation-no absorption of radiation, there will


not be fluorescence.
2. Rigidity of structures-Rigid structures will produce
more fluorescence,while flexible structure will
produce less fluorescence.
3. Nature of substituent groups-Electron donating
groups like amino, hydroxyl groups enhance
fluorescence activity,Electron withdrawing groups
reduce fluorescence. (groups like Nitro, carboxylic)
4.Viscosity-To increase fluorescence intensity increase the
viscosity (decrease collisions of molecules)
5.temperature-To increase fluorescence intensity decrease
the temperature (decrease collisions of molecules)
6.Oxygen-Oxygen decreases the Fluorescence intensity in 2
ways ,Oxidizes fluorescence and non fluorescence
substances.
7.Effect of pH- a.Aniline:Neutral or alkaline - shows visible
fluorescence,
Acidic in UV region only.
b.Phenols:Acidic- don't give fluorescence
Alkaline-gives good fluorescence
ADVANTAGES

• It’s one of the newer methods and its potentialities are still largely
unexplored.

• It also affects precision. Up to 1% can be achieved easily in


Flourimetric.

• The method is very sensitive and also possesses specificity because


there is a choice of wavelength notonly for the radiation emitted, but
also for the light which excites it.
LIMITATIONS
• Careful buffering is necessary as fluorescence intensity may be strongly
dependent.

• Ultraviolet light used for excitation may cause photo chemical changes or
destruction of the fluorescent molecule.

• The presence of dissolved oxygen may cause increased photo chemical


destruction.

• Traces of iodide and nitrogen oxides are efficient quenchers and therefore
interfere.
PRECAUTIONS

• Fluorescence analysis is especially applicable to trace substances, care must be


taken to eliminate contaminations of sample..
• Rubber and cork stoppers contain fluorescent materials and these are extracted
if the solvent touches them.
• Filter paper also contains fluorescent material which is extracted by solvents.
• Grease from stop cocks and other sources is a fluorescent contaminant.
• All glasses contain Al, Ca and SiO2 which may be extracted.
• The most important consideration is the concentration of the reagent.
Concentration must be expressed in micro molecules so that the ratio of the
reagent to metal may be estimated easily.
• Large temperature change between unknown and standard should be avoided.
• It is also not desirable to expose the solution to ultra violet radiation for longer
APPLICATIONS
1. APPLICATIONS IN INORGANIC CHEMISTRY
• Determination of ruthenium
• Determination of boron in steel
• Determination of aluminium in alloys
• Determination of chromium and manganese in steel
• Determination of uranium salts
• Estimation of rare earth terbium
• Estimation of bismuth
• Determination of beryllium in silicates
• Estimation of 3,4 benzpyrene
• Determination of zinc
• Determination of cadmium
APPLICATION IN ORGANIC CHEMISTRY

1. Assay of thiamine:
2. Estimation of quinine sulphate by fluorimetry
SPECIAL FLUORIMETRIC APPLICATIONS
1. Investigation of chemical structures and Processes
2. Chemical analysis
3. Laser induced fluorescence spectroscopy of human tissues for cancer diagn
4. Study of marine petroleum pollutants
5. Accurate determination of glucose
6. A highly sensitive fluorescent immunoassay based on avidin labeled nanocr
7. Flourescence polarisation immunoassay of mycotoxins
CONCLUSION
• Fluorescence spectroscopy is a sensitive optical emission technique in which
sample molecules are excited with a photon source.
• Those molecules that relax by radiant emission can be subsequently detected by
measuring the intensity of that emission.
• Fluorimetry is generally used if there is no colourimetric method sufficiently
sensitive or selective for the substance to be determined.
• The important applications for determination of organic and inorganic
compounds including immunoassays and chemistry of bioluminescence are
reviewed.
• Special fluorimetric applications are also included in this study.
REFERENCES
• Kommu Naresh, Applications of Fluorescence spectroscopy, Journal of
Chemical and Pharmaceutical sciences, [2014].
• Dr.S. Ravishankar, Text book of pharmaceutical analysis: Flurimetry,
edition 4.
• Douglas A Skoog, Donald M west, F James holler, Stanley R crouch,
Molecular fluorescence spectroscopy ,
• Text book of Fundamentals of analytical chemistry ,edition:8.
• BK Sharma, Instrumental methods of chemical analysis, Molecular
fluorescence spectroscopy [2011].
• AH Beckett, JB Stenlake, Practical pharmaceutical chemistry,
spectrofluorometry, part 2, edition: 4.
• Devala Rao, In practical pharmaceutical analysis, Estimation of
quinine sulphate by fluorimetry
• Arthus , Vogel, Text book of quantitative analysis. [8]. Chris
Maragos. Fluorescence Polarisation Immunoassay of Mycotoxins, A
Review, Journal of Toxins, [2009].
THANK YOU

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