FLUORIMETRY

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FLUORIMETRY FLOURIMETRY

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9895620706 DEPT. OF PHARM ANALYSIS AL SHIFA COLLEGE OF PHARMACY PERINTHALMANNA

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INTRODUCTION DEFINITION THEORY FACTORS AFFECTING FLOURESCENCE INSTRUMENTATION APPLICATIONS IN PHARMACY CONCLUSION REFERENCES

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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

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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 .

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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 .

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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.

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Ground excited singlet triplet state singlet state spins unpaired states spin paired no net mag.field net mag.field

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Fluorescence Phosphorescence Radiation less processes Vibration relaxation Internal conversion External conversion Intersystem crossing

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LIGHT EMITING AT ONCE SOURCE STARTS & STOPS WHEM SOURCE STOPS

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JABLONSKI ENERGY DIAGRAM

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FLUORESCENCE AND CHEMICAL STRUCTURE Fluorescence is most commonly observed in compounds containing aromatic functional groups with low energy. Most unsubstituted aromatic hydrocarbons show fluorescence - quantum efficiency increases with the no: of rings and degree of condensation.

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CONTD … Simple heterocyclic do not exhibit fluorescence. The n - *singlet quickly converts to the n - * triplet and prevents fluorescence.

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Fusion of heterocyclic nucleus to benzene ring increases fluorescence .

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Substitution on the benzene ring shifts wavelength of absorbance maxima and corresponding changes in fluorescence peaks Fluorescence decreases with increasing atomic no: of the halogen. Substitution of carboxylic acid or carboxylic group on aromatic ring inhibits fluorescence .

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Fluorescence is favored in molecules with structural rigidity. organic chelating agents complexed with metal ion increases fluorescence .

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Nature of molecule Nature of substituent Effect of concentration Adsorption, Light Oxygen,ph Photodecomposition Temp . &viscosity Quantum yield Intensity of incident light Path length

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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.

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nature of substituent Electron donating group enhances fluorescence – e.g.:NH 2 ,OH etc. Electron withdrawing groups decrease or destroy fluorescence. e.g.:COOH,NO 2 , N=N etc. High atomic no: atom introduced into  electron system decreases fluorescence.

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Fluorescence is directly proportional to concentration.

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FI = Q X I a i.e, F = QI O act Q = Constant for a particular substance I O = 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.

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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.

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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.

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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.

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K f = fluorescence k ec = external conversion k ic = internal conversion k isc = intersystem crossing k pd = pre dissociation K d = dissociation fluorescence quantum yield:

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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

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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

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Decrease in fluorescence intensity due to specific effects of constituents of the solution. Due to concentration, ph, pressure of chemical substances, temperature, viscosity, etc. Types of quenching Self quenching Chemical quenching Static quenching Collision quenching

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Fluorescence Concentration of fluorescing species Deviations at higher concentrations can be attributed to self-quenching or self-absorption . Fluorescence Concentration of fluorescing species Calibration curve (Low con) calibration curve (High con)

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Here decrease in fluorescence intensity due to the factors like change in ph,presence of oxygen, halides &heavy metals. ph - aniline at ph 5-13 gives fluorescence but at ph <5 &>13 it does not exhibit fluorescence. halides like chloride,bromide,iodide & electron withdrawing groups like no2,cooH etc. leads to quenching. Heavy metals leads to quenching, because of collisions of triplet ground state.

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This occurs due to complex formation. e.g.. caffeine reduces the fluorescence of riboflavin by complex formation. COLLISIONAL QUENCHING It reduces fluorescence by collision. where no. of collisions increased hence quenching takes place.

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Fluorescence mainly classified in to 2 categories. Based on wavelength of emitted radiation Stokes fluorescence Antistokes fluorescence Resonance fluorescence Based on phenomenon Sensitized fluorescence Direct line fluorescence Stepwise fluorescence Thermally assisted fluorescence.

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Based upon wavelength: stokes fluorescence: wavelength of emitted radiation is longer than absorbed radiation. Anti stokes: wavelength of emitted radiation is shorter than absorbed radiation. Resonance fluorescence: wavelength of emitted radiation is equal to absorbed radiation .

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Sensitized fluorescence- When elements like thalium,zn,cadmium or an alkali metal are added to mercury vapour these elements are sensitized and thus gives fluorescence . Direct line fluorescence Even after the emission of radiation, the molecules retain in metastable state and finally comes to the ground state after loss of energy by vibrational transmit.

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Stepwise fluorescence this is conventional type of fluorescence where a part of energy is lost by vibrational transition before the emission of fluorescent radiation. Thermally assisted fluorescence here excitation is partly by electromagnetic radiation and partly by thermal energy.

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INSTRUMENTATION

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SOURCE OF LIGHT FILTERS AND MONOCHROMATORS SAMPLE CELLS DETECTORS

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MERCURY ARC LAMP. XENON ARC LAMP. TUNGSTEN LAMP. TUNABLE DYE LASERS .

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MERCURY ARC LAMP Produce intense line spectrum above 350nm. High pressure lamps give lines at 366,405, 436, 546,577,691,734nm. Low pressure lamps give additional radiation at 254nm.

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Intense radiation by passage of current through an atmosphere of xenon. Spectrum is continuous over the range between over 250-600nm,peak intensity about 470nm .

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Intensity of the lamp is low. If excitation is done in the visible region this lamp is used. It does not offer UV radiation.

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Pulsed nitrogen laser as the primary source. Radiation in the range between 360 and 650 nm is produced.

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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.

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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.

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PHOTOVOLTAIC CELL PHOTO TUBE PHOTOMULTIPLIER TUBES – Best and accurate.

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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 10 6 is obtained.

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SINGLE BEAM FLUORIMETER DOUBLE BEAM FLUORIMETER SPECTROFLUORIMETER(DOUBLE BEAM)

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Tungsten lamp as source of light. The primary filter absorbs visible radiation and transmits uv radiation. Emitted radiation measured at 90 o by secondary filter. Secondary filter absorbs uv radiation and transmits visible radiation.

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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.

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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.

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Sample & reference solution can be analyzed simultaneously. disadvantage Rapid scanning is not possible due to use of filters.

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Power supply Source primary filter secondary filter Detector Sample cell Slit Data processor

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The primary filter in double beam fluorimeter is replaced by excitation monochromaters. The secondary filter is replaced by emission monochromaters. The incident beam is split into sample and reference beam using a beam splitter. The detector is photomultiplier tube.

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Advantages Rapid scanning to get Exitation & emission spectrum. More sensitive and accuracy when compared to filter fluorimeter .

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Powersupply Source Excitation monochromator Emission monochromator Detector Sample cell Data processor

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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 .

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Field determination of uranium salts. 3]fluorescent indicators Mainly used in acid-base titration. e.g.: eosin- colorless-green. Fluorescein:colourless-green. Quinine sulphate: blue-violet. Acridine: green-violet

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Reagent Ion Fluorescence wavelength Sensitivity Alizarin garnet B Al 3+ 500 0.007 Flavanol 8-Hydroxy quinoline Sn 4+ Li 2+ 470 580 0.1 0.2 4] Fluorometric reagent Aromatic structure with two or more donor functional groups

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compound reagent excitation wavelength fluorescence hydrocortisone 75%v/v H 2 SO 4 in ethanol 460 520 nicotinamide cyanogen chloride 250 430 5] organic analysis Qualitative and quantitative analysis of organic aromatic compounds present in cigarette smoke, air pollutants, automobile exhausts etc. 6] pharmaceutical analysis

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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.

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Fluorimetry ,nowadays can be used in detection of impurities in nanogram level better than absorbance spectrophotometer with special emphasis in determining components of sample at the end of chromatographic or capillary column.

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Douglas A Skoog, Principles of instrumental analysis H:\UV-Vis Luminescence Spectroscopy - Theory.mht Dr.B.K.Sharma, Instrumental methods of chemical analysis Gurdeep R Chatwal, Instrumental methods of chemical analysis

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http://images.google.co.in/imghp?oe=UTF-8&hl=en&tab=wi&q=fluorescence http://en.wikipedia.org/wiki/Fluorescence http://www.bertholdtech.com/ww/en pub/bioanalytik/biomethods/fluor.cfm