SPECTROFLUORIMETRY 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. .

LUMINESCENCE SPECTROSCOPY Pharmaceutical Analysis.. .

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. .

Energy Level Diagram     .

SPECTROFLOURIMETER

INSTRUMENTATION

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.

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.

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

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.

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

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

4. Collisional Quenching 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. Conjugation Nature of Substituent group Rigidity of structure Effect of Temperature Viscosity Oxygen Effect of Ph Effect of Concentration

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

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)

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.

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 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