Ima Presentation

7/30/2019 Ima Presentation

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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.JPG


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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=AFQjCNEZN6gMnqYvvoPV6j3O2TVE8y2t8A


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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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