POGONATUM : morphology, anatomy, reproduction etc.
Spectroflourimetry.pdf
1. SHREE CHANKYA EDUCATION SOCIETY,
INDIRA COLLEGE OF PHARMACY,PUNE
Seminar on
1
Presented by : Mr.Namit
S.Tajane
First Year M.Pharm
(Pharmaceutics)
Roll No.17
Guided by,
Dr. Manasi Wagdarikar
Department : Analytical
Chemistry
2. Contents:
1. Introduction
2. Principle
3. Jablonski Energy Diagram
4. Spectrofluorometer
5. Components of Spectrofluorometer
6. Application of Spectrofluorometry
7. Research Paper
8. References
2
3. Introduction
Spectroscopy: Spectroscopy is study of interaction between matter and
electromagnetic Radiation.
• The matter can be Atoms, Molecules or Ions.
• When the electromagnetic Radiation interact with matter then it can
either absorb or emit energy.
• This emission and absorption can be takes place at both molecular and
atomic level.
3
4. Understanding the terms…
• Singlet Ground state: State in which electrons in a molecule are paired[↑↓]
• Singlet excited state: State in which electrons are unpaired but of opposite spins.
• Triplet state: State in which unpaired electrons of same spin are present[↑↑]
• Excitation process : Absorption of Energy or light followed by conversion from
ground state to excite state
• Relaxation Process : Process by which atom or molecule losses energy & returns
to ground state.
4
6. Principle :
• Spectrofluorimetry or Fluorescence spectroscopy is a technique in
which UV/ Visible light is absorbed by electrons in singlet ground state
to move to singlet excited state and returns back to the singlet ground
state by emitting radiation (fluorescence) with lower energy (lower
wavelength) or same energy to give rise to fluorescence spectroscopy.
6
• Excitation fluorescence Spectrum
• Emission fluorescence Spectrum
8. Flouresence Phosphorescence
• Radiation is emitted only when
light incident on them
• It involve immediate emission of
light
• It does not involve change in
direction of spin
• The time interval between
absorption and emission is very
short
• Radiation is emitted even after
the light source is removed
• It involve delayed emission of
light
• It involve the change in direction
of Spin(Singlet to Triplet)
• The time interval between
absorption and emissiom is long
8
11. Sources : In Spectroflourimeter xenon Arc lamp as a source of Radiation
It produce more intense radiation than mercury arc lamp and gives continuous
spectrum between 250 and 660 nm.
Excitation Monochromator :
Allows only the excitation wavelength one at a time and absorbs all the other
wavelength and this happens for the complete chosen range of wavelength e.g.
Diffraction grating
Sample Cell (Cuvette) : Sample is placed in it and the cell is polished on all
the four sides, as the fluorescence emission is measured at 90 degree to the
incident light e.g. Quartz.
11
12. 12
Emission/Fluorescence monochromator : Allows only the emission
Wavelength one at a time and absorb all the other wavelengths and this
happens for the complete chosen range of wavelength e.g. Diffraction
grating.
Detector : It detects the intensity of the fluorescence light for each
wavelength and generates current proportional to it e.g.Photomultiplier
Recorder : It records
1. Excitation fluorescence spectrum –
2. Emission fluorescence spectrum -
13. What can Spectrofluorometer Do?
• It is has been used for the direct or indirect Quantitative and Qualitative analysis
by Measuring the fluorescent intensity F
• Effect of concentration on fluorescence intensity :
• Fluorescence Intensity = Q x Ia
Where, Ia = Io x act,
Io= Intensity of incident light
So, Fluorescence Intensity = Q x Io x act
Where, Q = Fluorescence efficiency
Ia= Intensity of absorbed light
a= Molecular Extinction Coefficient
t = Path length
c = Concentration of substance 13
15. Quenching of Fluorescence :
• Quenching refers to the any process that reduces the fluorescence
intensity of a given Substance.
• This may occurs due to Various factors like pH, Concentration,
temperature, viscosity, Presence of oxygen.
• Example of Quenching agents:
15
Quenching Agent Typical fluorophore
Thiocynate Anthracene
Chloride Quinine
Iodide Tryptophan
Disulfide Tyrosine
Nitric oxide Naphthalene
16. Factors Responsible for Quenching:
1. Temperature of Solution
2. Viscosity of the solution
3. Polarity and pH of solution
4. Presence of any paramagnetic species in solution
5. Concentration of the solution
6. Self absorption
16
17. Application of Spectroflourimetry
Determination of Organic substances:
Plant pigment, steroids ,proteins, Napthols etc can be determined at low
Concentration. Generally used to carry out qualitative as well as
quantative analysis for a great aromatic compounds present in cigarette
smoking ,air pollutant concentrates & automobiles exhaust.
Determination of inorganic Substance:
• Extensively used in the field of nuclear research for the determination
of uranium salt.
• Determination of vitamin B both 1 and 2
• Most important application are found in the analyses of Food products,
Pharmaceuticals, clinical sample and natural products.
17
18. 18
Fluorescent indicators:-
Intensity and colour of the fluorescence of many substances depend upon
the pH of solutions. These are called as fluorescent indicators and are
generally used in acid base titrations.
Eg: 1. Eosin - pH 3.0-4.0 - colourless to green
2. Fluorescein - pH 4.0-6.0 - colourless to green
21. 21
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 colorimetric 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.
Conclusion :
22. References :
1. Skoog ,Holler and Crouch, “Principles of Instrumental Analysis”6thedition-
2007 books/cole,part of cengage learning,page no. 407
2. Aswathy Bose, Irene Thomas, Kavitha G and Elessy Abraham/ International
Journel of Advances in Pharmaceutical analysis 2018;08(01):01-08.
3. https://www.slideshare.net/jobinkv/spectrofluorimetry
4. https://www.youtube.com/watch?v=W9p6Y8IW89A
5. .Dr. Supriya S. Mahajan ,Instrumental Methods Of Analysis, popular prakashan,
page no.155.
22
