Fluorimetry: Molecular Fluorescence Spectroscopy PDF

FLOURIMETRY DR. AFSHAN ABDUL SHAKOOR  CONTENTS  INTRODUCTION  DEFINITION  THEORY  FACTORS AFFECTING FLOURESCENCE  INSTRUMENTATION  APPLICATIONS IN PHARMACY  Introduction  Flourimetry is the measurement of fluorescence at a particular wavelength w...

FLOURIMETRY DR. AFSHAN ABDUL SHAKOOR  CONTENTS  INTRODUCTION  DEFINITION  THEORY  FACTORS AFFECTING FLOURESCENCE  INSTRUMENTATION  APPLICATIONS IN PHARMACY  Introduction  Flourimetry is the measurement of fluorescence at a particular wavelength with the help of spectrofluorimeter.  Luminescence is the emission of light by a substance. It occurs when an electron returns to the electronic ground state from an excited state and loses its excess energy as a photon.  It is of 3 types. Fluorescence spectroscopy. Phosphorescence spectroscopy. Chemiluminescence spectroscopy  Fluorescence  When a beam of light is incident on certain substances they emit visible light or radiations. This is known as fluorescence.  Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off.  The substances showing this phenomenon are known as flourescent substances.  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 called phosphorescence. Substances showing phosphorescence are phosphorescent substances  Theory of Fluorescence AND Phosphorescence A molecular electronic state in which all of the electrons are paired are called singlet state. In a singlet state molecules are diamagnetic. Most of the molecules in their ground state are paired. When such a molecule absorbs uv/visible radiation, one or more of the paired electron raised to an excited singlet state /excited triplet state Ground excited singlet Triplet state singlet state spins unpaired states spin paired no net mag.field net mag.field LIGHT EMITING AT ONCE SOURCE STARTS & STOPS WHEM SOURCE STOPS Nature of molecule Nature of substituent Effect of concentration Adsorption, Light Oxygen,ph  Photodecomposition Temp. &viscosity Quantum yield Intensity of incident light Path length  Nature of molecules All the molecules cannot show the phenomenon of fluorescence. Only the molecules absorbs UV/visible radiation can show this phenomenon. Greater the absorbency of the molecule the more intense its fluorescence.  Nature of substituent Electron donating group enhances fluorescence – e.g.:NH2,OH etc. Electron withdrawing groups decrease or destroy fluorescence. e.g.:COOH,NO2, N=N etc. High atomic no: atom introduced into  electron Fluorescence is directly proportional to concentration.  FI = Q X Ia i.e, F = QIOact Q = Constant for a particular substance IO = Constant for an instrument a = Molecular extinction coefficient t = Path length C = Concentration of the substance F = KC Where K represents all constants FI α Concentration.  Extreme sensitiveness of the method requires very dilute solution. Adsorption of the fluorescent substances on the container wall create serious problems. Hence strong solutions must be diluted.  LIGHT Monochromatic light is essential for the excitation of fluorescence because the intensity will vary with wavelength. OXYGEN The presence of oxygen may interfere in 2 ways. 1] by direct oxidation of the fluorescent substances to non fluorescent. 2] by quenching of fluorescence.  PH Alteration of the ph of the solution will have significant effect on fluorescence. Fluorescent spectrum is different for ionized and un-ionized species. TEMPERATURE & VISCOSITY Increase in temperature/decrease in viscosity will decrease fluorescence. Increase in intensity of light incident on sample Increases fluorescence intensity. The intensity of light depends upon 1)light emitted from the lamp. 2)Excitation monochromaters. 3)Excitation slit width The effective path length depends on both the excitation and emission slit width. Use of microcuvette does not reduce the fluorescence. Use of microcell may reduce interferences and increases the measured fluorescence  QUENCHING Decrease in fluorescence intensity due to specific effects of constituents of the solution. Due to concentration, ph, pressure of chemical substances, temperature, viscosity, etc INSTRUMENTATION  Components of spectroflourimeter SOURCE OF LIGHT FILTERS AND MONOCHROMATORS SAMPLE CELLS DETECTORS  Source of light MERCURY ARC LAMP. High pressure lamps give lines at 366, 405, 436, 546, 577, 691, 734nm. Low pressure lamps give additional radiation at 254nm. XENON ARC LAMP. Spectrum is continuous over the range between over 250- 600nm,peak intensity about 470nm. TUNGSTEN LAMP. If excitation is done in the visible region this lamp is used. It does not offer UV radiation. TUNABLE DYE LASERS  FILTERS Primary filter-absorbs visible light & transmits uv light. Secondary filter-absorbs uv radiations & transmits visible light. MONOCHROMATORS Exitation monochromaters-isolates only the radiation which is absorbed by the molecule. Emission monochromaters-isolates only the radiation emitted by the molecule. The majority of fluorescence assays are carried out in solution. Cylindrical or rectangular cells fabricated of silica or glass used. Path length is usually 10mm or 1cm. All the surfaces of the sample holder are polished in fluorimetry. PHOTOVOLTAIC CELL PHOTO TUBE PHOTOMULTIPLIER TUBES – Best and accurate. Multiplication of photo electrons by secondary emission of radiation. A photo cathode and series of dynodes are used. Each cathode is maintained at 75-100v higher than the preceding one. Over all amplification of 106 is obtained. Tungsten lamp as source of light. The primary filter absorbs visible radiation and transmits uv radiation. Emitted radiation measured at 90o by secondary filter. Secondary filter absorbs uv radiation and transmits visible radiation. Simple in construction Easy to use. Economical disadvantages It is not possible to use reference solution & sample solution at a time. Rapid scanning to obtain Exitation & emission spectrum of the compound is not possible. Similar to single beam instrument. Two incident beams from light source pass through primary filters separately and fall on either sample or reference solution. The emitted radiation from sample or reference pass separately through secondary filter.  Sample & reference solution can be analyzed simultaneously. Disadvantage  Rapid scanning is not possible due to use of filters.  Sample Power Sourc primary filter cell supply e Slit secondary filter Detector Data processor 1] Determination of inorganic substances Determination of ruthenium ions in presence of other platinum metals. Determination of aluminum (III) in alloys. Determination of boron in steel by complex formed with benzoin. Estimation of cadmium with 2-(2 hydroxyphenyl) benzoxazole in presence of tartarate. Field determination of uranium salts. 3]fluorescent indicators Mainly used in acid-base titration. e.g. Fluorescein:colourless-green. Quinine sulphate: blue-violet. Acridine: green-violet  4] Fluorometric reagent  Aromatic structure with two or more donor functional groups Reagent Ion Fluorescen Sensitivi ce ty wavelengt h Alizarin Al3+ 500 0.007 garnet B Flavanol Sn4+ 470 0.1 8- Li2+ 580 0.2 Hydroxy 5] Organic Analysis Qualitative and quantitative analysis of organic aromatic compounds present in cigarette smoke, air pollutants, automobile exhausts etc. 6] Pharmaceutical Analysis compound reagent excitation fluorescence wavelengt h hydrocortiso 75%v/v 460 520 ne H2SO4 in ethanol nicotinamide cyanoge 250 430 n chloride 7] Liquid chromatography Fluorescence is an imp method of determining compounds as they appear at the end of chromatogram or capillary electrophoresis column. 8]Determination of vitamin B1 &B2.