(11) Fluorimetry Theory | Concept of Singlet, Doublet, Triplet state, Internal & External Conversion

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Fluorimetry is a powerful analytical technique used to detect and quantify substances based on their fluorescent properties. It involves the emission of light by a substance that has absorbed light or electromagnetic radiation at a different wavelength. Here's an overview of the theory behind fluorimetry:
Fluorescence and Fluorescent Molecules
Excitation and Emission: Fluorescence occurs when a molecule, called a fluorophore, absorbs photons of light at a specific wavelength (excitation wavelength). The absorbed energy promotes electrons to higher energy levels. Shortly after, the electrons return to their ground state, emitting light at a longer wavelength (emission wavelength).
Stokes Shift: The emitted light typically has a longer wavelength and lower energy than the absorbed light due to energy losses through various relaxation processes within the molecule. This difference between the excitation and emission wavelengths is called the Stokes shift.
Fluorimeter Components and Operation
Light Source: Fluorimeters use a light source, often a xenon or mercury lamp, that emits light across a range of wavelengths. The light is directed through a monochromator or filters to select the desired excitation wavelength.
Sample Chamber: The sample containing the fluorophore is placed in a sample chamber where it's exposed to the selected excitation wavelength.
Detector: A detector measures the emitted fluorescent light from the sample. A monochromator or filter can be used to select the specific emission wavelength to be measured.
Data Analysis: The emitted light is quantified by the detector, and the resulting data can be analyzed to determine the concentration or properties of the fluorescent substance. Calibration curves relating fluorescence intensity to known concentrations are often used for quantitative analysis.
Applications of Fluorimetry
1. Quantitative Analysis: Determining the concentration of fluorescent molecules in a sample by measuring the emitted light intensity.
2. Biosensing: Used extensively in biological research, including DNA analysis, protein assays, and cell imaging.
3. Environmental Monitoring: Detecting and quantifying pollutants or environmental contaminants.
Medical Diagnostics: Assessing various biomarkers and disease states based on fluorescence properties.
Factors Affecting Fluorescence
Several factors can influence fluorescence measurements:
1. Quenching: Processes that reduce fluorescence intensity, such as collisions with other molecules or impurities.
2. Photobleaching: Continuous exposure to light leading to the irreversible destruction of the fluorophore.
3. pH and Temperature: Changes in these factors can affect the fluorescence properties of the molecule.
4. Solvent Effects: The nature of the solvent can impact the fluorescence behavior of the molecule.
Fluorimetry's sensitivity and selectivity make it a valuable tool in various scientific fields, allowing for precise analysis and detection of fluorescent compounds in diverse samples.
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