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Prepared By:
HIMANSHU SHARA2010B2A2196P
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THEORY Spectrophotometry is a method of analyzing involving
how light interacts with the atoms (or molecules) in asample of matter.
Visible light is only a small portion of the entireelectromagnetic spectrum and it includes the colorscommonly observed (red, yellow, green, blue andviolet).
The visible spectrum consists of electromagneticradiation of wavelengths ranging from 400 nm tonearly 800 nm.
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INTRODUCTION A large no. of substances which absorb U.V. or
Visible light energy loss excess energy as heat
through collision with neighboring atom or molecule. However a few number of these substances loss
only a part of this excess energy as heat and emit the
remaining energy as electromagnetic radiation of
longer wavelength than that of absorbed. This processof emitting radiation is known as
LUMISNESCENCE.
.
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.
LUMINESCENCE
FLUORESCENCE
PHOSPHORESCENCEPECTROSCOPY
CHEMILUMINESCENCE
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Energy Level Diagram
.
s2
SINGLET STATES TRIPLET STATES
Ground
State
s1
T
T
1
2
INTERSYSTEM
CROSSING
VIBRATIONAL
RELAXATION
FLUORESCENCE PHOSPHORESCENCE
INTERNAL
CONVERSION CONVERSION
INTERNAL
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SPECTROFLOURIMETER
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INSTRUMENTATION
http://en.wikipedia.org/wiki/File:Spectrophotomer.JPG7/30/2019 Ima Presentation
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ADVANTAGES SENSITIVITY : It is more sensitive as concentration
is low as g/ml or ng/ml.
PRECISION : Upto 1 % can be achieved. SPECIFICITY : More specific than absorption method
where absorption maxima may be same for twocompounds.
RANGE OF APPLICATION : Even non fluorescentcompounds can also be converted to fluorescentcompounds by chemical compounds.
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SOLVENT EFFECT
Solvents characteristics have important effects onfluroescent behavior of molecules.
Three main effects can be recognized: A. Solvent Polarity
A polar solvent is preferred as the energy requiredfor the * is lowered. B. Solvent Viscosity
More viscous solvents are preferred since collisionaldeactivation will be lowered at higher viscosities.
C. Heavy Atoms Effect
If solvents contain heavy atoms, fluorescencequantum efficiency will decrease andphosphorescence will increase.
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DETERMINATION OF UNKNOWN
CONCENTRATION BY FLUORIMETRY.
The fluorescence intensity is proportional to concentration
From Lambert-Beers law
F = 2.303 Iocl.......................................... (A)
where F= Intensity of fluorescent radiation
= Fluorescence quantum yield
Io= Intensity of incident radiation
http://www.google.co.in/url?sa=t&rct=j&q=fluorescence%20quantum%20yield%20symbol&source=web&cd=1&ved=0CCQQFjAA&url=http://goldbook.iupac.org/Q04991.html&ei=Z4ykUIn2A6SaiAe_rYGQCQ&usg=AFQjCNEZN6gMnqYvvoPV6j3O2TVE8y2t8Ahttp://www.google.co.in/url?sa=t&rct=j&q=fluorescence%20quantum%20yield%20symbol&source=web&cd=1&ved=0CCQQFjAA&url=http://goldbook.iupac.org/Q04991.html&ei=Z4ykUIn2A6SaiAe_rYGQCQ&usg=AFQjCNEZN6gMnqYvvoPV6j3O2TVE8y2t8Ahttp://www.google.co.in/url?sa=t&rct=j&q=fluorescence%20quantum%20yield%20symbol&source=web&cd=1&ved=0CCQQFjAA&url=http://goldbook.iupac.org/Q04991.html&ei=Z4ykUIn2A6SaiAe_rYGQCQ&usg=AFQjCNEZN6gMnqYvvoPV6j3O2TVE8y2t8Ahttp://www.google.co.in/url?sa=t&rct=j&q=fluorescence%20quantum%20yield%20symbol&source=web&cd=1&ved=0CCQQFjAA&url=http://goldbook.iupac.org/Q04991.html&ei=Z4ykUIn2A6SaiAe_rYGQCQ&usg=AFQjCNEZN6gMnqYvvoPV6j3O2TVE8y2t8A7/30/2019 Ima Presentation
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= molar absorptivity
l = path lengthc = concentration of analyte
Fluorescence intensity (F) is directly proportional to the
concentration (of analyte), and intensity of incident radiation (I o)
But, when concetration increases, fluorescence does not
increase proportionally because self quenching or
concentration quenching comes in picture.
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CONCENTRATION AND FLUORESCENCE
INTENSITY
F
Conc. of fluorescing species
c1
For aconcentration
above c1 thecalibration curveis no longerlinear.
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Quenching
It is a decrease in flourescence intensity due to specific effectsof constituents of the solution like concentration, pH,temperature, viscosity etc.
Type of Quenching
1. Self or Concentration Quenching
Low conc.-Increase in flourescence intensity(g/ml or ng/ml) linearity observed.
High conc.- Decrease in flourescence intensity
(mg/ml)
2. Chemical Quenching
Oxygen Halide & electron withdrawing group
Heavy metal
PH
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3. Static Quenching
This occur because of complex formation.
4. Collisional Quenching
Halide
Heavy metal
Increase in temperature
Decrease in viscosity
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Factors affecting
Fluorescence Intensity. Conjugation
Nature of Substituent group
Rigidity of structure Effect of Temperature
Viscosity
Oxygen
Effect of Ph
Effect of Concentration
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1.Conjugation
Subtituent Effect on intensity
alkyl Increase
COOH, CHO, COOR Decrease
OH, NH2, NHR, NR2 Increase
F, Cl, Br, I Decrease
Molecule must have unsaturation to give flourescence.
2. Nature of Substituent group
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3. Rigidity of structure Rigid structure More flourescence intensity
Flexible structure - Less flourescence intensity4. Viscosity
Increase Viscosity Increase flourescence intensity
5. Effect of Concentration Low conc.-Increase flourescence intensity
(g/ml or ng/ml)
High conc.- Decrease flourescence intensity
(mg/ml)
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6. Effect of Temperature
From 25-60C in 5C increments.
Higher temperatures result in larger collisional deactivation due to
increased movement and velocity of molecules. Therefore, lowertemperatures are preferred.
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7. Oxygen
It oxidies flourescent substance to non- flourescent
substance
8. Effect of pH
Aniline in alkaline medium gives visible flourescence
& in acidic medium gives flourescence in UV regiononly.
Phenol in acidic medium- No flourescence
in alkaline medium- Gives flourescence
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1. Determination of polyaromatic hydrocarbons
Benzo[a]pyrene is a product of incomplete combustion and
found in coal tar.
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2. Determination of Organic substances:-
Aromatic polycyclicHydrocarbons
Indoles
NaphtholsProteins
Steroids
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3. List of drugs that can be assayed by Flourimetry
Drug Excitation
wavelength(nm)
Emission
wavelength(nm)
P-aminosalisylic acid 300 404
Amphotericin 340 421
Chlordiazepoxide 285 510
Cyclizine 305 417
Diphenhydarmine 305 412
Fluphenazine 290 480
Flourazepam 375 475
Oxazepam 365 490
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