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FLOURIMETRY- Principle,Procedure & uses.
- 1. FLOURIMETRY Prepared by Ms. G.Sowmya B.Pharm 4th Year Ratnam Institute of Pharmacy Under the guidance of Dr.M.Suchitra, Professor & HOD, Department of Pharmaceutical Chemistry &Analysis Ratnam Institute of Pharmacy
- 2. Contents INTRODUCTION DEFINITION THEORY FACTORS EFFECTING FLOURESCENCE QUENCHING INSTRUMENTATION APPLICATIONS CONCLUSION
- 3. INTRODUCTION Flourimetry isa process in which sample concentration is determined by measuring the intensity of flouriscence & phosphorescence. Fluorimetry is a photometric technique used in chemical analysis. It measures the fluorescence emitted by substances when they are excited by ultraviolet or visible light. This method is widely used in drug analysis, biochemistry, and environmental science because of its high sensitivity and selectivity. Types of fluorescence: Flourescence Phosphorescence
- 4. FLOURESCENCE Definition: Fluorescence isthe spontaneous emission of light by a substance that has absorbed light or electromagnetic radiation. It occurs when electrons in a molecule absorb energy, move to an excited state, and then quickly return to the ground state by releasing energy in the form of visible light It is a instantaneous process. It starts soon after light absorption and stops as soon as the incident light is cut off.
- 5. PHOSPHORESCENCE Definition: Phosphorescence isa type of photoluminescence where a substance absorbs light and emits it slowly over a longer period, even after the light source is removed. Unlike fluorescence, phosphorescence involves a triplet excited state, where electrons undergo a forbidden transition back to the ground state. As a result, the emission is delayed and can last from microseconds to several minutes or hours. Also called delayed flourescence.
- 6. THEORY FLOURESCENCE & PHOSPHORESCENCE A molecular electronic state in which all electrons are paired is called a singlet state. In a singlet state, molecules are diamagnetic.Most molecules in their ground state exist in the singlet state. When such a molecule absorbs UV or visible radiation, one or more of the paired electrons get raised to an excited singlet state/excited triplet state.
- 7. Concepts of singlet,doubletand triplet states These are electronic states that describe the number of unshared pairs of electrons present in a molecule. Electrons are present in pair ↑↓.
- 8. PRINCIPLE When abeam of light is passed through a sample,molecule of sample absorb get converted into singlet ground state to single excited state. When molecules get backing to their ground state they emit the light know as flourescence.
- 9. INTERNAL & EXTERNALCONVERSIONS INTERNAL CONVERSIONS: It is an intermolecular process which brings down a molecules to a lower energy electronic state without emitting light. It involves vibrational relaxation in singlet excited state,singlet excited state to triplet excited state and vibrational relaxation in triplet state. Time:10 ¹¹ to 10 . ⁻ ⁻⁹ EXTERNAL CONVERSION: It is an process which is molecules brings down it’s energy to electronic state by emitting light. It involves singlet excited state to singlet ground state and triplet excited state to singlet ground state with emission of light.
- 10. FACTORS EFFECTING FLOURESCENCE Flourescenceintensity is affected by two factors: 1. Structural Factors: Conjugation Nature of substituent group Rigidity of structure 2. Non-structural Factors: Temperature Viscosity Oxygen Concentrationy
- 11. STRUCTURAL FACTORS Conjugation: oMolecule should be unsaturated for fluorescence. o Conjugation is directly proportional to fluorescence intensity.( = bonds, lone pairs → higher fluorescence). Nature of Substituent Group: o Electron-withdrawing groups (e.g., NO , COOH) decrease fluorescence intensity. ₂ o These groups cause shortening of conjugation. o Electron-donating groups (e.g., NH , OH) increase fluorescence intensity. ₂ Rigidity of the Structure: o Rigid structures show higher fluorescence intensity(e.g., fluorine).
- 12. NON STRUCTURAL FACTORS Temperature: o Increase in temperature increases molecular collisions and decreases fluorescence intensity. Viscosity: o Inversely proportional to fluorescence. Oxygen: o Inversely proportional to fluorescence.Oxygen oxidizes the molecules and converts them into non-fluorescent substances. Concentration: o Directly proportional to fluorescence intensity, but not in every case. o It depends on the intensity of incident light. pH: o pH affects fluorescence intensity but varies from compound to compound.Example: Imidazole. o Fluorescence is inversely proportional to pH.
- 13. QUENCHING It isa process by which the intensity of fluorescence decreases.It is due to the effect of the sample. Types of Quenching: 1. Concentration Quenching: o Occurs when the solution absorbs an excess amount of primary or fluorescent radiation.it is also called an inner filter effect (due to collisions). 2. Chemical Quenching: o In this, the intensity of fluorescence decreases due to changes in the chemical nature of the solution (e.g., decomposition). o Example: Aniline gives fluorescence at pH between 5–13. 3. Static Quenching: o It is due to the formation of a complex, which decreases fluorescence intensity. 4. Collision Quenching: o Increase in collision between molecules decreases fluorescence intensity.
- 14. INSTRUMENTATION 1. Lightsource 2.Filters/monochromaters 3.Sample cells 4.Detector 5.Read out system
- 15. LIGHT SOURCE Highintensity radiant energy should be supplied from the light source,as flourescence is directly proportional to intensity. Commonly used light sources: Laser lamp Deuterium lamp Mercury vapour lamp Xenon lamp
- 16. FILTERS Filters basicallyabsorb the incident light of undesired wavelength and passed light of desired wavelength. Act like slits. TYPES OF FILTERS: 1.Primary filter: o Passes incident light to the sample. o They take visible light and passes to detector. 2.secondary filters: o Get light from the sample and passes to detector. o They take uv light and passes visible light.
- 17. SAMPLE CELLS Theyhold samples like glass cuvetts,quartz cuvetts,matched cuvetts,etc.
- 18. DETECTORS Same asuv visible spectro photo meter mostly use photo multiplier tube detector.
- 19. APPLICATIONS Determination ofinorganic substances i.e. Al, Li, etc. Determination of thiamine HCl. Determination of Phenytoin. Determination of Indole, phenols, phenothiazines. Determination of proteins, plant pigments.
- 20. CONCLUSION Fluorimetry isa sensitive technique used for detecting and estimating fluorescent substances. It is based on the principle of fluorescence emission after absorbing light. This method is fast, accurate, and suitable for both qualitative and quantitative analysis. It is widely used in pharmaceutical, clinical, and environmental fields. Despite some limitations, it remains an important analytical tool in modern science.