(12) Fluorimetry | Factors Affecting Fluorescence, Quenching | Instrumental Methods of Analysis

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Quenching in fluorescence refers to any process that reduces the intensity of fluorescence emission from a fluorophore. It can occur due to various mechanisms and factors, impacting the accuracy and reliability of fluorescence measurements. Here are some key factors affecting fluorescence quenching:
Factors Contributing to Quenching:
Collisional Quenching: This occurs when the excited fluorophore interacts with a quencher molecule through collisions. The energy from the excited state can transfer to the quencher, reducing fluorescence. It can be dynamic (temporary) or static (resulting in a non-fluorescent complex).
Oxygen: Molecular oxygen is a common quencher as it can interact with the excited state of the fluorophore, leading to the loss of fluorescence. This process is known as dynamic or collisional quenching due to oxygen's high reactivity.
Temperature: Higher temperatures can increase molecular motion, leading to more collisions between the fluorophore and potential quenchers. This can enhance quenching effects.
pH: Changes in pH can alter the environment around the fluorophore, affecting its fluorescence. Some quenchers are more effective at specific pH ranges.
Heavy Metals and Ions: Certain metal ions or heavy metals can act as quenchers by interacting with the fluorophore and altering its electronic structure, thus affecting its fluorescence.
Concentration of Quencher: An increased concentration of quencher molecules can result in higher rates of collisional quenching, leading to decreased fluorescence intensity.
Proximity of Quencher to Fluorophore: The spatial proximity of the quencher to the fluorophore plays a crucial role. Quenching is more likely to occur if the quencher is in close proximity to the excited state fluorophore.
Methods to Minimize Quenching:
Controlled Environment: Ensure the sample is prepared in an environment with controlled factors such as temperature, pH, and oxygen levels to minimize quenching effects.
Choosing Appropriate Fluorophores: Select fluorophores less susceptible to quenching by certain ions, molecules, or environmental factors.
Use of Protective Agents: Employ substances or conditions that can shield the fluorophore from quenching agents. For instance, adding certain chemicals can create a protective environment.
Dilution: Diluting the sample can decrease the concentration of quenchers, thereby reducing the chances of collisional quenching.
Understanding the various factors contributing to quenching is crucial in designing experiments, optimizing conditions, and interpreting fluorescence data accurately in fluorimetry assays and analyses.
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[@ajaykumar-wt3fo]

Temp is inversely proportional to the fluorescence intensity

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