Fluorescence Spectroscopy: Principles, Techniques, and Applications
- 1. Under the supervision of :- Dr. Santosh Kumar Associate Professor Department of Biotechnology Guru Ghasidas Vishwavidyalay, Bilaspur (C.G.) FLUORESCENT SPECTROSCOPY Presented By :- Bhavika Venkata Lkshmi Cheshtarani Yadav Dharun Sao Ehtesham Raza Firdous Ashraf
- 3. INTRODUCTION Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscop y that analyzes fluorescence from a sample.
- 4. INTRODUCTION Fluorescence spectroscopy analyzes fluorescence from a molecule based on its fluorescent properties. Fluorescent spectroscopy is a powerful analytical technique used to study the fluorescent properties of substances. It involves exciting a molecule with a specific wavelength of light and measuring the emitted light at a longer wavelength. This method is widely used in various scientific fields, including chemistry, biology, medicine, and materials science, due to its high sensitivity and specificity.
- 5. INTRODUCTION • Fluorescence spectroscopy is a fast, simple and inexpensive method to determine the concentration of an analyte in solution based on its fluorescent properties. • It can be used for relatively simple analyses, where the type of compound to be analyzed ('analyte') is known, to do a quantitative analysis to determine the concentration of the analytes. • Fluorescence is used mainly for measuring compounds in
- 6. PRINCIPLE When a molecule absorbs light of a specific wavelength (energy), it transitions from the ground electronic state to an excited electronic state . 1.Excitation After excitation, the molecule loses energy through non-radiative processes (e.g., vibrational relaxation or internal conversion). This energy loss means that the molecule typically relaxes to the lowest vibrational level of the excited state before emitting light. 2.Relaxation Fluorescence spectroscopy is based on the absorption of light by a substance and the subsequent emission of light at a longer wavelength. This process involves transitions between electronic energy levels in a molecule and can be summarized in the 3.Fluorescence Emission From the lowest vibrational level of the excited state, the molecule emits light as it transitions back to the ground state. The emitted light has lower energy (longer wavelength) than the absorbed light, a phenomenon known as the Stokes shift.
- 7. ENERGY DIAGRAM It is a schematic representation of the transition of electronic state of a molecule during the fluorescence phenomenon. The left axis shows increasing energy, where a typical fluorescent molecule has an absorbance spectrum. This spectrum shows the energy or wavelengths, where the molecule will absorb light.. The process can be visualized with a Jablonski diagram, which depicts the energy levels of a molecule and the transitions during absorption, relaxation, and emission.
- 8. KEY FEATURES High Sensitivity Selectivity Fluorescence spectroscopy can detect even minute quantities of fluorescent substances due to the amplification of the signal through the emission of light. Each fluorophore has distinct excitation and emission wavelengths, allowing for selective detection of specific molecules in complex mixtures.
- 9. KEY FEATURES Structural Information Molecular Environment: The fluorescence spectrum is sensitive to the molecular environment of the fluorophore, providing information about factors like pH, polarity, and the presence of specific ions. Conformational Changes: Changes in the conformation of biomolecules can alter their fluorescence properties, allowing for the study of protein folding, DNA hybridization, and other structural changes.
- 10. KEY FEATURES Non-Destructive Analysis Minimal Sample Preparation: Fluorescence spectroscopy often requires minimal sample preparation, making it suitable for delicate samples and in-situ measurements. Preservation of Sample Integrity: The non-destructive nature of the technique allows for repeated measurements and further analysis.
- 11. MATHEMATICAL REPRESENTATION: Fluorescence lifetime of a molecule is the average length of time it spends in the excited state. k: Instrumental constant Ø: Quantum yield (efficiency of fluorescence emission) I : Intensity of the excitation light C: Concentration of the fluorescent species
- 12. Other Considerations Solvent Effects: The polarity and viscosity of the solvent can influence the fluorescence properties of a molecule. Temperature Effects: Temperature can affect the rate of non-radiative decay processes, influencing the fluorescence intensity and lifetime. pH Effects: The pH of the solution can affect the ionization state of the fluorophore, altering its absorption and emission properties.
- 13. By understanding these mathematical relationships and considering the various factors that can influence fluorescence, researchers can effectively utilize fluorescence spectroscopy to study a wide range of chemical and biological systems. Reference : https://wikivisually.com/wiki/Alloprotein
- 14. APPLICATIONS Biological Sciences • Protein Analysis • DNA and RNAAnalysis • Cellular Imaging Environmental Science • Water Quality Monitoring • Soil Analysis
- 15. APPLICATIONS Food Science • Food Quality Control Materials Science • Polymer Characterization • Semiconductor Analysis
- 16. ADVANTAGES AND LIMITATION Advantages Limitation High sensitivity Interference from other compounds Selectivity Photobleaching Versatility Matrix effects
- 17. CONCLUSION Fluorescence spectroscopy, a powerful analytical technique, has revolutionized various fields. By harnessing the principles of light absorption and emission, it enables us to probe the molecular structure, concentration, and interactions of analytes with remarkable sensitivity and specificity. From biomedical research to environmental monitoring, fluorescence spectroscopy continues to be an indispensable tool, driving innovation and expanding our understanding of the world around us.
- 18. Lakowicz, J. R. (2006). Principles of Fluorescence Spectroscopy. Springer. REFERENCE Valeur, B. (2002). Molecular Fluorescence: Principles and Applications. Wiley- VCH. Harris, D. C. (2010). Quantitative Chemical Analysis. W. H. Freeman and Company.
- 19. THANK YOU