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Fluorimetry: Theory

Fluorimetry is a type of spectroscopy concerned with the measurement of fluorescence from a sample.
In the context of pharmaceutical instrumental methods of analysis, fluorimetry is used to quantify the concentration of certain active pharmaceutical ingredients (APIs) or other molecules of interest in a sample, based on their fluorescent properties.

Here's a brief breakdown:

1. Principle:

When certain molecules absorb light (photons) of a specific wavelength, they become excited to a higher energy state.
As they return to their ground state, they release the excess energy in the form of emitted light, which is called fluorescence.
The intensity of this emitted light is often directly proportional to the concentration of the fluorescing molecule in the sample.

Excitation Source: Often a lamp emitting ultraviolet (UV) or visible light, used to excite the molecules in the sample.
Monochromator: Helps in selecting a specific wavelength of light to excite the sample.
Sample Cell: Where the sample is placed.
Emission Monochromator: After the sample has been excited, this selects a specific wavelength of emitted light to measure.
Detector: Measures the intensity of the emitted light.

Quantification: Measure the concentration of drug substances that are naturally fluorescent.
Analysis of Complex Mixtures: Fluorescent tagging can be used to make non-fluorescent molecules detectable.
Kinetic Studies: Monitor the rate of chemical reactions by observing changes in fluorescence.
Detection of Impurities: Trace amounts of fluorescent impurities can be detected, which is especially useful when these impurities might not be detectable using other methods.

High Sensitivity: Capable of detecting very low concentrations of compounds.
Selectivity: Due to the unique excitation and emission wavelengths of different compounds.

Not all compounds are naturally fluorescent.
Fluorescence can be affected by various factors, including pH, temperature, and presence of quenching agents.