Prepared By:

HIMANSHU SHARA 2010B2A2196P

THEORY
Spectrophotometry is a method of analyzing involving how light interacts with the atoms (or molecules) in a sample of matter. Visible light is only a small portion of the entire electromagnetic spectrum and it includes the colors commonly observed (red, yellow, green, blue and violet). The visible spectrum consists of electromagnetic radiation of wavelengths ranging from 400 nm to nearly 800 nm.

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 process of emitting radiation is known as LUMISNESCENCE.
.

FLUORESCENCE

LUMINESCENCE SPECTROSCOPY

PHOSPHORESCENCE CHEMILUMINESCENCE

Fluorescence
When a beam of light is incident on certain substances, they emit visible light or radiations. This phenomenon is known as Fluorescence

Phosphorescence
When light radiation is incident on certain substances, they emit light continuously even after the incident light is cut off. This type of delayed fluorescence is known as phosphorescence.

Energy Level Diagram

TRIPLET STATES

s2

VIBRATIONAL RELAXATION

T2 T1
INTERSYSTEM CROSSING PHOSPHORESCENCE INTERNAL CONVERSION INTERNAL CONVERSION

s1

FLUORESCENCE

Ground State

SPECTROFLOURIMETER

INSTRUMENTATION

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 two compounds. RANGE OF APPLICATION : Even non fluorescent compounds can also be converted to fluorescent compounds by chemical compounds.

SOLVENT EFFECT
Solvents characteristics have important effects on fluroescent behavior of molecules. Three main effects can be recognized:
A. Solvent Polarity

A polar solvent is preferred as the energy required for the * is lowered.

More viscous solvents are preferred since collisional deactivation will be lowered at higher viscosities.
C. Heavy Atoms Effect

If solvents contain heavy atoms, fluorescence quantum efficiency will decrease and phosphorescence will increase.

DETERMINATION OF UNKNOWN CONCENTRATION BY FLUORIMETRY.

F = 2.303 Io cl
..........................................

(A)

where F= Intensity of fluorescent radiation = Fluorescence quantum yield

Io= Intensity of incident radiation

= molar absorptivity l = path length c = 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.

CONCENTRATION AND FLUORESCENCE INTENSITY

c1 Conc. of fluorescing species

For a concentration above c1 the calibration curve is no longer linear.

Quenching
It is a decrease in flourescence intensity due to specific effects of 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

3. Static Quenching
This occur because of complex formation.

4. Collisional Quenching
Halide Heavy metal Increase in temperature Decrease in viscosity

Factors affecting Fluorescence Intensity.

Effect of Temperature
Viscosity Oxygen

Effect of Ph
Effect of Concentration

1.Conjugation
Molecule must have unsaturation to give flourescence.

2. Nature of Substituent group

Effect on intensity
Increase Decrease Increase Decrease

3. Rigidity of structure
Rigid structure More flourescence intensity Flexible structure - Less flourescence intensity

4. 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)

6. Effect of Temperature
Higher temperatures result in larger collisional deactivation due to increased movement and velocity of molecules. Therefore, lower temperatures are preferred.

From 25-60C in 5C increments.

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 region

only. Phenol in acidic medium- No flourescence in alkaline medium- Gives flourescence

APPLICATIONS.
1. Determination of polyaromatic hydrocarbons

Benzo[a]pyrene is a product of incomplete combustion and

found in coal tar.

2. Determination of Organic substances:Aromatic polycyclic Hydrocarbons Indoles Naphthols Proteins Steroids

3. List of drugs that can be assayed by Flourimetry

P-aminosalisylic acid Amphotericin Chlordiazepoxide Cyclizine

Diphenhydarmine
Fluphenazine Flourazepam

305
290 375

412
480 475

Oxazepam

365

490