Fluorometry (fluorescence and Phosphorescence Study), Slides of Pharmaceutical Analysis

Download Fluorometry (fluorescence and Phosphorescence Study) and more Slides Pharmaceutical Analysis in PDF only on Docsity! ~ > -Flouro ar a Aw + a e P 7 AN % - a ‘ Vv a - S Qe F V é fi \ metry y ry N e a e v4 nen c Prepared by: ns Runa Akter bd oy _ Lecturer, Uap as ar as Crs Bd Fluoresecnce Fluorescence is a spectrochemical method of analysis where the molecules of the analyte are excite by irradiation at a certain wavelength and emit radiation of a different wavelength. The emission spectrum provides information for both qualitative and quantitative analysis. When certain chemical substances are excited electronically by absorption of UV or visible radiation, they emit light at a longer wave length. This phenomenon is called luminescence. Depending on the life span of the excited species, two different processes could be distinguished. these are fluorescence and phosphorescence. Fluorescence: When the luminescence stops within 10-8 to 10-4 sec after the source of excitation is removed, it is called fluorescence. Phosphorescence: When the luminescence continues for a slightly longer period of time (»10-4 to 10 sec) after the source of excitation is removed, it is called phosphorescence. Molecules in the ground state At room temp molecules reside in ground state. The ground electronic state is usually a singlet state in which all of the electrons are paired and in each pair the two electrons spin about their axis in opposite direction. The ground state can be subdivided into many states termed as vibration energy level. Excitation and distribution of molecules in excited state Due to absorption of UV and visible light, molecules are transmitted in the excited electronic state. Excited electronic state is also subdivided into various vibrational energy level and excited molecules will be distributed in various vibrational energy levels of excited state. Excited state is also known as excited singlet state. Resonance fluorescence 6 Molecules from the exited singlet state can loss energy by several mechanism & return to the ground state. If the excited molecules emit radiation at the same wavelength, the process is termed as resonance fluorescence which is a very rare process. Vibrational relaxation Rather molecules undergo a radiation less loss of vibrational energy and quickly fall to the lower vibrational energy level of the excited state. The vibrational energy is thought to be lost to solvent molecules. The process is known as vibrational relaxation. Fluorescence From the lowest vibrational energy level of the excited state, electrons can return to the ground state by photo emission and this process is known as fluorescence. Because of the vibrational relaxation, the radiation emitted as fluorescence is of lower energy & therefore, of longer wavelength than that originally absorbed. 7 Internal conversion An excited molecule may also loss energy by other processes. For example, it may undergo a radiation less loss of energy sufficient to drop to the ground state. The process is known as internal conversion. This is a competitive process of fluorescence. Fluorescence vs Phosphorescence Fluorescence Phosphorescence Definition Definition It ceases as soon as the source of excitation radiation has been removed Phosphorescence can continue after the source of excitation radiation has been removed Energy of emitted radiation is relatively higher Energy of emitted radiation is relatively lower It is used as analytical tool. It is not used as analytical tool Fluorometry VS spectrophotometry Factors Fluorometry Spectrophotometry Sensitivity Fluorometry is more sensitive as an analytical tool than is spectrophotometry. Spectrophotometry is less sensitive than Fluorometry. Specificity It is more specific in identifyingand analyzing a compound than spectrophotometry. It is less specific compared to fluorometry. Temperatur e Intensity of fluorescence may decrease with the increase oftemperature. Small variation of temperaturedoes not affect much. pH Intensity of fluorescence is pH dependent, especially for the solutions of weak acids and weak bases. Small variation of pH does notaffect much. Intensity of incident light For a given concentration, the intensity of fluorescence mayincrease by increasing the intensity of the incident light. Absorbance is independent of theintensity of the incident light. Stability of incident light Intensity of incident light must be stable. Intensity of incident light is not strictly controlled. Fluorometer / Spectrofluorometer Any instrument used to measure the intensity of fluorescence is known as fluorometer or spectrofluorometer. The design and function of a fluorometer are quite similar to those of a spectrophotometer. The chief components of a fluorometer are- • Radiation sources • Excitation filter or monochromator • Sample holder • Emission filter or monochromator • Detector • Recorder Factors influencing the intensity of fluorescence ▸ Concentration of fluorescing species, ▸ Presence of other solutes as impurity, ▸ pH of the solution, ▸ Temperature of the solution, ▸ Degradation/Stability of sample, ▸ Solvent effects, etc. Relation between concentration of fluorescing species and fluorescence intensity: The intensity of fluorescence is directly proportional to the amount of light absorbed by the sample solution. F α (Io-I)…………..(i) Io=intensity of incident light I= intensity of transmitted light. Again, intensity of fluorescence is directly proportional to quantum efficiency of fluorescence (φ) F α φ……….(ii) From equation (i) and (ii) we can write F α φ (Io-I) Or F =K φ (Io-I)……(iii) From Beer’s law I=Io e -Ebc E=Molar absorptivity B=path length of irradiation C=conc. in moles per litre Now from equation (iii) we can write F =K φ (I0-Io e –Ebc) = K φ Io (1- e –Ebc)…….(iv) At low concentration, the intensity of fluorescence is directly proportional to the conc. of the sample solution. At high conc. e –Ebc is about to zero, so at high conc. the intensity of fluorescence is independent of conc. Of the sample solution. Relation between fluorescence intensity and conc. Chemical quenching Quenching may be defined as a process in which the chemicals reduce the observed fluorescence by the formation of complex with fluorescent molecule either in ground state or excited state. And the chemical are called Quencher. Chemical quenching is of two tyoes: ▸ Collision quenching ▸ Static quenching Collision quenching In this case, quencher binds with the fluorescent molecule in excited state. This interaction results in the dissipatiion of excited energy not by fluorescence but by transfer of energy to the quencher. Static quenching Here, quencher binds with the fluorescent molecule in ground state and inhibits the molecule to go to excited state and thus inhibits to show fluorescence. Relation between fluorescence and chemical structure There are some structural features which influence the fluorescence process of organic compounds. They are discussed below: Compound having conjugated double bond: In this case degree of fluorescence depends on • Degree of conjugation • Delocalization of pi electrons (resonance) Saturated compound (having no conjugated double bond) such cyclohexane is non- fluorescent, while benzene is weakly fluorescent die to resonance and highly unsaturated polycyclic compounds are strongly fluorescent. Substituted benzene compound Mono substitution of benzene compound: mono substitution of benzene compound with alkyl group have little influent on fluorescence intensity. Mono substation of benzene compound with ortho-para direction group (-NH2, -OH, OCH3,-NHR, NR2) Increase fluorescence intensity than parent compound due to depolarization of pi electrons. Exception: (Cl-, Br-, I- o-p directing group but their substituted benzene compounds are either not fluorescent or less fluorescent than benzene. Due to intersystem crossing process, they show intense phosphorescence but only weak fluorescence. Meta directing: Most meta directing groups (-NH2, -CHO, -COOH) markedly decrease fluorescence intensity of benzene compounds since they tend to localize pi electrons. Exception: Though (-C=N) nitrile group is meta directing of benzene compounds, it increases fluorescence intensity. Disubstitution of Aromatic ring: the fluorescence characteristics of disubstituted benzenes are unpredictable. For example, substitution of fluorescent compound (aniline) with a meta directing group such as –SO2NH2 should decrease the fluorescent intensity. But actually sulfanilamide is 5 times more fluorescent then aniline. Application of Fluorometry Trace amount of impurities can be detected by fluorometric method. Chemical Application: Fluorometric technique can be used in inorganic chemistry for • Determination of metal ions • Filtration • Separation and identification Biopharmaceutical application: Fluorometric technique can be used in bio-pharmaceutics for • Measurement of drug in blood, urine and other fluids • Study of rate and mechanism of drug absorption metabolism and excretion • Selection of toxic compound Pharmaceutical application: the technique is used for quantitative analysis of following drugs and drug classes- Hormone: such as- adrenaline, aldosterone, testosterone Alkaloids • Opium alkaloids-Morphine, codeine etc. • Rauwolfia alkaloids-reserpine. • Others-Atropine, emetine etc. Vitamins: such as thiamine (B1) , Riboflavin (B2) Antibiotics: such as tetracycline, sulfonamide etc. Cardiac glycoside: such as digoxin, Digitoxin etc. fluorometric technique can also be used for qualitative analysis of drug.