1. SPECTROFLUORIMETRY
Prepared By:
HIMANSHU SHARA
2010B2A2196P

2. 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.

3. 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. .

4. LUMINESCENCE SPECTROSCOPY
Pharmaceutical Analysis.. .

5. Fluorescence Phosphorescence
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. .

6. Energy Level Diagram
 
  .

7. SPECTROFLOURIMETER

8. INSTRUMENTATION

9. ADVANTAGES
SENSITIVITY : It is more sensitive as concentration is low as µg/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.

10. 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 π−π* is lowered.
B. Solvent Viscosity
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.

11. DETERMINATION OF UNKNOWN CONCENTRATION BY FLUORIMETRY.
The fluorescence intensity is proportional to concentration
From Lambert-Beer’s law
F = 2.303Φ Io εcl
(A)
where F= Intensity of fluorescent radiation
Φ = Fluorescence quantum yield
Io= Intensity of incident radiation

12. ε = 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.

13. CONCENTRATION AND FLUORESCENCE INTENSITY
For a concentration above c1 the calibration curve is no longer linear.

14. Quenching Type of Quenching 1. Self or Concentration 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
(µg/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

15. 4. Collisional Quenching
3. Static Quenching
This occur because of complex formation.
4. Collisional Quenching
Halide
Heavy metal
Increase in temperature
Decrease in viscosity

16. Factors affecting Fluorescence Intensity.
Conjugation
Nature of Substituent group
Rigidity of structure
Effect of Temperature
Viscosity
Oxygen
Effect of Ph
Effect of Concentration

17. 2. Nature of Substituent group
1.Conjugation
Molecule must have unsaturation to give flourescence.
2. Nature of Substituent group
Subtituent
Effect on intensity
alkyl
Increase
COOH, CHO, COOR
Decrease
OH, NH2, NHR, NR2
F, Cl, Br, I

18. 5. Effect of Concentration
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
(µg/ml or ng/ml)
High conc.- Decrease flourescence intensity
(mg/ml)

19. 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 25-60ºC in 5ºC increments.

20. 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

21. APPLICATIONS. 1. Determination of polyaromatic hydrocarbons
Benzo[a]pyrene is a product of incomplete combustion and found in coal tar.

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

23. 3. List of drugs that can be assayed by Flourimetry
Excitation wavelength(nm)
Emission wavelength(nm)
P-aminosalisylic acid
300
404
Amphotericin
340
421
Chlordiazepoxide
285
510
Cyclizine
305
417
Diphenhydarmine
412
Fluphenazine
290
480
Flourazepam
375
475
Oxazepam
365
490