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5. Specroscopy.pptx Dr Thirunahari Ugandhar

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Head Department of Botany Govt Degree College Mahabubaba
Head Department of Botany Govt Degree College Mahabubaba

Dr Thirunahari Ugandhar

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Spectroscopy UV-Vis Spectroscopy & Fluorescence Spectroscopy Dr. Thirunahari Ugandhar Associate Prof of Botany Department of Botany Kakatiya Govt College (A) Hanamkonda
UV-Vis Spectroscopy • What is UV-Vis Spectroscopy? UV-Visible spectroscopy (UV- Vis) is an analytical technique that examines the interaction of ultraviolet (200–400 nm) and visible light (400–800 nm) with matter. • It measures the amount of light absorbed or reflected by a sample, providing insights into its composition and concentration. • This method is widely used due to its simplicity, cost- effectiveness, and versatility
• Ultraviolet-Visible (UV-Vis) Spectrophotometer A UV-Vis spectrophotometer is a device designed to analyze materials such as liquids, films, powders, and solids. It works by passing light through a sample and measuring the amount absorbed or transmitted. • • Wavelength Range: UV-Vis spectrophotometers operate within 200–800 nm, covering both ultraviolet and visible light regions. • • Light Sources: These instruments use specific light sources: • Deuterium Lamp: Covers the UV range. • Tungsten-Halogen Lamp: Extends into the visible spectrum. • Both lamps ensure full coverage of the wavelength range.
• Theory Behind UV-Vis Spectroscopy UV-Vis spectroscopy relies on the interaction of light with a sample. When light interacts with the electron cloud in a chromophore (light-absorbing part of a molecule), it excites electrons from a lower-energy state to a higher-energy state. This process results in an absorption spectrum that can be analyzed to identify or quantify substances. • Key points: • Absorption Spectrum: Represents how much light a sample absorbs at specific wavelengths. • Beer’s Law: Relates absorbance to concentration, enabling quantitative measurements. • Chromophore Analysis: Chromophores absorb specific wavelengths, providing a fingerprint for identification.
• Applications of UV-Vis Spectroscopy UV-Vis spectroscopy is a versatile tool with applications spanning various fields: • 1. Quantitative Analysis • Used to measure concentrations of analytes in solutions, such as drugs, food additives, and environmental samples. • Integrated with High-Performance Liquid Chromatography (HPLC) and Ultra- High-Performance Liquid Chromatography (UHPLC) for enhanced detection. • 2. Research and Development • Analyzes trace metal content, e.g., manganese in steel. Metals are reacted to form measurable complexes, like manganate (VII). • Identifies optical properties of materials such as thin films and nanoparticles. • 3. Quality Control • Ensures consistency in manufacturing processes by verifying product composition. • Common in pharmaceutical and industrial laboratories. • 4. Educational and Fundamental Studies • Demonstrates principles like Beer’s Law and molecular absorption in academic settings. • 5. Environmental and Regulatory Monitoring • Measures pollutants and water quality indicators, such as nitrate or phosphate levels.
• Advantages of UV-Vis Spectroscopy • Non-Destructive: Samples remain intact during analysis.Versatile: Suitable for a wide range of organic and inorganic compounds. • Cost-Effective: Affordable compared to other spectroscopic methods. • Fast and Accurate: Provides rapid results with high precision. • UV-visible spectroscopy is a cornerstone analytical technique, invaluable in research, industry, and academia because it provides quick and reliable insights into materials' chemical and physical properties.
5. Specroscopy.pptx Dr Thirunahari Ugandhar
5. Specroscopy.pptx Dr Thirunahari Ugandhar
Fluorescence Spectroscopy • Introduction: Fluorescence spectroscopy, also known as fluorometry or spectrofluorimetry, is a branch of electromagnetic spectroscopy used to analyze fluorescence emitted by a sample. It employs ultraviolet (UV) light to excite electrons in molecules, causing them to emit light of lower energy, typically in the visible spectrum. A complementary method to fluorescence spectroscopy is absorption spectroscopy. The devices used for fluorescence measurement are referred to as fluorometers or fluorimeters
• Principles of Fluorescence Spectroscopy • Molecules exhibit various energy states, classified as electronic and vibrational levels. Fluorescence spectroscopy primarily focuses on the interaction of light with these states. • 1. Photon Absorption: Molecules absorb photons, causing their electrons to transition from a ground state to an excited state. • 2. Vibrational Relaxation: Collisions with surrounding molecules lead to a loss of vibrational energy, settling the molecule into the lowest vibrational state of the excited electronic level. • 3. Photon Emission: The molecule returns to its ground state, emitting photons with energies corresponding to different vibrational levels.
• Key Spectra • • Emission Spectrum: Measured by holding the excitation wavelength constant while recording the intensity of fluorescence across various wavelengths. • • Excitation Spectrum: Obtained by varying the excitation wavelength while recording the total emitted fluorescence at a fixed wavelength.
• Instrumentation • 1. Types of Instruments: • Filter Fluorometers: Use filters to isolate excitation and emission light. • Monochromators: Allow precise selection of wavelengths for excitation and emission, enhancing versatility. • 2. Detectors: • Single-Channel Detectors: Measure one wavelength at a time. • Multi-Channel Detectors: Record intensities at all wavelengths simultaneously, eliminating the need for monochromators. • 3. Light Sources: • Continuous light sources, like xenon or mercury lamps, are used for steady excitation across a broad spectrum.
5. Specroscopy.pptx Dr Thirunahari Ugandhar
Fluorescence Spectroscopy:
5. Specroscopy.pptx Dr Thirunahari Ugandhar
5. Specroscopy.pptx Dr Thirunahari Ugandhar
• Data Analysis • At low concentrations, fluorescence intensity is directly proportional to the concentration of the fluorophore. However, achieving accurate spectra requires addressing several factors: • 1. Instrument-Related Factors: • Lamp Intensity Variability: Light source intensity can fluctuate over time. • Monochromator and Filter Efficiency: Transmission efficiency may vary with wavelength. • Detector Quantum Efficiency: The proportion of photons detected depends on the detector's specifications and conditions. • 2. Sample-Related Factors: • Photodecomposition: Fluorophores may degrade upon prolonged exposure to excitation light. • Scattering: • Rayleigh Scattering: Light scattered at the same wavelength as the incident light. •  Raman Scattering: Scattered light shifts to longer wavelengths due to molecular interactions. • Inner Filter Effects: •  Reabsorption: Emitted photons are reabsorbed by other molecules, distorting the spectrum. •  High Concentration Effects: Uneven distribution of excitation light within the sample can reduce fluorescence intensity.
• Applications of Fluorescence Spectroscopy • 1. Biochemical and Medical Research: • Detection and quantification of biomolecules like proteins, DNA, and RNA. • Differentiation of malignant and benign tumors, including skin cancer studies. • 2. Chemical Analysis: • Analysis of organic compounds and trace elements in various solutions. • 3. Environmental Studies: • Monitoring pollutants and studying complex organic interactions in ecosystems. • 4. Pharmaceutical Industry: • Characterization of drug formulations and active compounds.
• Fluorescence spectroscopy is a vital tool in research and industry, offering a sensitive, non-destructive method for analyzing complex molecular interactions and properties. While challenges such as instrument variability and inner filter effects exist, advancements in instrumentation and analytical techniques continue to enhance its precision and application scope.
5. Specroscopy.pptx Dr Thirunahari Ugandhar
5. Specroscopy.pptx Dr Thirunahari Ugandhar

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5. Specroscopy.pptx Dr Thirunahari Ugandhar

  • 1. Spectroscopy UV-Vis Spectroscopy & Fluorescence Spectroscopy Dr. Thirunahari Ugandhar Associate Prof of Botany Department of Botany Kakatiya Govt College (A) Hanamkonda
  • 2. UV-Vis Spectroscopy • What is UV-Vis Spectroscopy? UV-Visible spectroscopy (UV- Vis) is an analytical technique that examines the interaction of ultraviolet (200–400 nm) and visible light (400–800 nm) with matter. • It measures the amount of light absorbed or reflected by a sample, providing insights into its composition and concentration. • This method is widely used due to its simplicity, cost- effectiveness, and versatility
  • 3. • Ultraviolet-Visible (UV-Vis) Spectrophotometer A UV-Vis spectrophotometer is a device designed to analyze materials such as liquids, films, powders, and solids. It works by passing light through a sample and measuring the amount absorbed or transmitted. • • Wavelength Range: UV-Vis spectrophotometers operate within 200–800 nm, covering both ultraviolet and visible light regions. • • Light Sources: These instruments use specific light sources: • Deuterium Lamp: Covers the UV range. • Tungsten-Halogen Lamp: Extends into the visible spectrum. • Both lamps ensure full coverage of the wavelength range.
  • 4. • Theory Behind UV-Vis Spectroscopy UV-Vis spectroscopy relies on the interaction of light with a sample. When light interacts with the electron cloud in a chromophore (light-absorbing part of a molecule), it excites electrons from a lower-energy state to a higher-energy state. This process results in an absorption spectrum that can be analyzed to identify or quantify substances. • Key points: • Absorption Spectrum: Represents how much light a sample absorbs at specific wavelengths. • Beer’s Law: Relates absorbance to concentration, enabling quantitative measurements. • Chromophore Analysis: Chromophores absorb specific wavelengths, providing a fingerprint for identification.
  • 5. • Applications of UV-Vis Spectroscopy UV-Vis spectroscopy is a versatile tool with applications spanning various fields: • 1. Quantitative Analysis • Used to measure concentrations of analytes in solutions, such as drugs, food additives, and environmental samples. • Integrated with High-Performance Liquid Chromatography (HPLC) and Ultra- High-Performance Liquid Chromatography (UHPLC) for enhanced detection. • 2. Research and Development • Analyzes trace metal content, e.g., manganese in steel. Metals are reacted to form measurable complexes, like manganate (VII). • Identifies optical properties of materials such as thin films and nanoparticles. • 3. Quality Control • Ensures consistency in manufacturing processes by verifying product composition. • Common in pharmaceutical and industrial laboratories. • 4. Educational and Fundamental Studies • Demonstrates principles like Beer’s Law and molecular absorption in academic settings. • 5. Environmental and Regulatory Monitoring • Measures pollutants and water quality indicators, such as nitrate or phosphate levels.
  • 6. • Advantages of UV-Vis Spectroscopy • Non-Destructive: Samples remain intact during analysis.Versatile: Suitable for a wide range of organic and inorganic compounds. • Cost-Effective: Affordable compared to other spectroscopic methods. • Fast and Accurate: Provides rapid results with high precision. • UV-visible spectroscopy is a cornerstone analytical technique, invaluable in research, industry, and academia because it provides quick and reliable insights into materials' chemical and physical properties.
  • 9. Fluorescence Spectroscopy • Introduction: Fluorescence spectroscopy, also known as fluorometry or spectrofluorimetry, is a branch of electromagnetic spectroscopy used to analyze fluorescence emitted by a sample. It employs ultraviolet (UV) light to excite electrons in molecules, causing them to emit light of lower energy, typically in the visible spectrum. A complementary method to fluorescence spectroscopy is absorption spectroscopy. The devices used for fluorescence measurement are referred to as fluorometers or fluorimeters
  • 10. • Principles of Fluorescence Spectroscopy • Molecules exhibit various energy states, classified as electronic and vibrational levels. Fluorescence spectroscopy primarily focuses on the interaction of light with these states. • 1. Photon Absorption: Molecules absorb photons, causing their electrons to transition from a ground state to an excited state. • 2. Vibrational Relaxation: Collisions with surrounding molecules lead to a loss of vibrational energy, settling the molecule into the lowest vibrational state of the excited electronic level. • 3. Photon Emission: The molecule returns to its ground state, emitting photons with energies corresponding to different vibrational levels.
  • 11. • Key Spectra • • Emission Spectrum: Measured by holding the excitation wavelength constant while recording the intensity of fluorescence across various wavelengths. • • Excitation Spectrum: Obtained by varying the excitation wavelength while recording the total emitted fluorescence at a fixed wavelength.
  • 12. • Instrumentation • 1. Types of Instruments: • Filter Fluorometers: Use filters to isolate excitation and emission light. • Monochromators: Allow precise selection of wavelengths for excitation and emission, enhancing versatility. • 2. Detectors: • Single-Channel Detectors: Measure one wavelength at a time. • Multi-Channel Detectors: Record intensities at all wavelengths simultaneously, eliminating the need for monochromators. • 3. Light Sources: • Continuous light sources, like xenon or mercury lamps, are used for steady excitation across a broad spectrum.
  • 17. • Data Analysis • At low concentrations, fluorescence intensity is directly proportional to the concentration of the fluorophore. However, achieving accurate spectra requires addressing several factors: • 1. Instrument-Related Factors: • Lamp Intensity Variability: Light source intensity can fluctuate over time. • Monochromator and Filter Efficiency: Transmission efficiency may vary with wavelength. • Detector Quantum Efficiency: The proportion of photons detected depends on the detector's specifications and conditions. • 2. Sample-Related Factors: • Photodecomposition: Fluorophores may degrade upon prolonged exposure to excitation light. • Scattering: • Rayleigh Scattering: Light scattered at the same wavelength as the incident light. •  Raman Scattering: Scattered light shifts to longer wavelengths due to molecular interactions. • Inner Filter Effects: •  Reabsorption: Emitted photons are reabsorbed by other molecules, distorting the spectrum. •  High Concentration Effects: Uneven distribution of excitation light within the sample can reduce fluorescence intensity.
  • 18. • Applications of Fluorescence Spectroscopy • 1. Biochemical and Medical Research: • Detection and quantification of biomolecules like proteins, DNA, and RNA. • Differentiation of malignant and benign tumors, including skin cancer studies. • 2. Chemical Analysis: • Analysis of organic compounds and trace elements in various solutions. • 3. Environmental Studies: • Monitoring pollutants and studying complex organic interactions in ecosystems. • 4. Pharmaceutical Industry: • Characterization of drug formulations and active compounds.
  • 19. • Fluorescence spectroscopy is a vital tool in research and industry, offering a sensitive, non-destructive method for analyzing complex molecular interactions and properties. While challenges such as instrument variability and inner filter effects exist, advancements in instrumentation and analytical techniques continue to enhance its precision and application scope.